(12) United States Patent (10) Patent No.: US 9.289,396 B2

US009289.396 B2

DeVore et al. 45) Date of Patent: Mar. 22,9 2016

(54) COLLAGEN-BASED IMPLANTS FOR A647/42 (2006.01) SUSTAINED DELVERY OF DRUGS A619/00 (2006.01) A647/46 (2006.01) (75) Inventors: Dale P. Devore, Chelmsford, MA (US); A 6LX3/55.75 (2006.01) Bruce H. Dewoolfson, Vienna, VA (US); (52) U.S. Cl. Eliot Lazar, Orchard Park, NY (US) CPC ...... A6IK 9/7007 (2013.01); A61 K9/0024 (2013.01); A61 K9/0051 (2013.01); A61 K9/06 (73) Assignee: Euclid Systems Corporation, Herndon, (2013.01); A61 K3I/55.75 (2013.01); A61 K VA (US) 47/42 (2013.01); A61 K47746 (2013.01) (*)c Notice:- r Subject to any disclaimer,- the term of this (58) CPCField ...... of Classification A61 K9/06; SearchA61 K47/46; A61K 3 1/5575 patent is extended or adjusted under 35 See application file for complete search histo U.S.C. 154(b) by 352 days. pp p ry. (56) References Cited (21) Appl. No.: 13/813,557 U.S. PATENT DOCUMENTS (22) PCT Filed: Aug. 1, 2011 5,219,895 A * 6/1993 Kelman ...... CO9J 189/06 (86). PCT No.: PCT/US2O11?046098 522.68 5,259.998 A * 1 1/1993 Reich et al...... 264/1.1 S371 (c)(1), 5,874,537 A 2f1999 Kelman et al. 6,261,547 B1 * 7/2001 Bawa et al...... 424/78.04 (2), (4) Date: Jan. 31, 2013 2009/028O158 A1 11/2009 Butuner et al. (87) PCT Pub. No.: WO2012/018715 OTHER PUBLICATIONS PCT Pub. Date: Feb. 9, 2012 International Search Report from the US Patent Office for Interna tional Application No. PCT/US2011/046098, mailed Jan. 10, 2012. (65) Prior Publication Data US 2013/0129807 A1 May 23, 2013 k cited. by examiner Primary Examiner — Ali Soroush Related U.S. Application Data (74) Attorney, Agent, or Firm — Finnegan, Henderson, (60) Provisional application No. 61/369.996, filed on Aug. Farabow, Garrett & Dunner LLP 2, 2010. (57) ABSTRACT (51) Int. Cl. A.tly provides collagen-based constructs for use in A6 IK9/06 (2006.01) A 6LX 9/70 (2006.01) 17 Claims, 3 Drawing Sheets U.S. Patent Mar. 22, 2016 Sheet 1 of 3 US 9.289,396 B2

FIG 1

160.00 - wn omorror- ex-wn -W------

l 140.00 8 120.00 - 3. t 100.00

n 80.00 (SRS8Set 1 O 33%;Set 2 p 60.00 r 40.00 S t 20.00

O.OO ------I day 0 day 3 day 4 day 8 day 10 day 14 day 17 day 21 U.S. Patent Mar. 22, 2016 Sheet 2 of 3 US 9.289,396 B2

5000 ------

p 4500 8 4000

3500

3000

2500

2000 1500 A Series1

1OOO

500

day day day day day day day 10 30 50 75 100 150 180

Release from non-pretreated Latanoprost Wafers U.S. Patent Mar. 22, 2016 Sheet 3 of 3 US 9.289,396 B2

2500

2000

1500

1000

500

Collection Day

Release from pretreated Latanoprost Wafers US 9,289,396 B2 1. 2 COLLAGEN-BASED IMPLANTS FOR entire human body). The same delivery issues exist in con SUSTAINED DELVERY OF DRUGS nection with tissues leading into the inner ear (e.g. the round window membrane). RELATED APPLICATIONS Conventional methods for delivery of therapeutic agents to the inner ear involve filling the middle ear with a solution or This application claims benefit of U.S. Provisional Appli other carrier of the therapeutic agent. Although these methods cation No. 61/369.996, filed Aug. 2, 2010, the disclosure of may ultimately result in delivery of drug into the inner ear which is incorporated by reference in its entirety. (e.g., by perfusion through the round window membrane), delivery of the therapeutic agent is generally not well con FIELD OF THE INVENTION 10 trolled and/or use of the carrier materials may be associated with adverse side effects. The present invention describes collagen-based constructs Thus, there is also a need for a sustained, controlled deliv for Sustained delivery of agents and drugs to a localized site of ery system for otic agents. interest. Sinus (Nasal) Tissues 15 Similar to ocular and otic liquid treatments, there are issues regarding nasal treatments. Treatments for sinusitis include BACKGROUND systemic antibiotics, but systemic administration of antibiot ics, particularly over extended periods as may be required to Oral or injectable drugs are commonly used to treat various treat sinusitis, can have undesirable effects on the flora of the diseases and conditions. However, these therapeutic digestive tract and reproductive system. Intranasal corticos approaches result in Systemic drug exposure that may be teroid sprays and intranasal decongestant sprays and drops unnecessary or even undesirable. Further, most orally-admin have also been used. However, the use of intranasal sprays istered drugs require at least daily dosing to maintain and drops by most patients does not result in the drug actually adequate drug levels. Even when targeted drug delivery is entering the affected intranasal sinuses. Rather, Such sprays possible because of the accessible location of the tissue to be 25 and drops typically contact only tissues located within the treated, it is still often difficult to attain adequate drug levels nasal cavity. over an extended period of time for various reasons. Some of Therefore, there is also a need for a sustained, controlled these difficulties are illustrated by treatments targeted to the delivery systems for sinus (nasal) agents. eyes, ears, and nasal sinuses. Drug Delivery Systems Ocular Tissues 30 Various methods have been developed to prolong drug The treatment of many ophthalmic diseases and post-op exposure following a single dosing. For example, the drug erative conditions require frequent administration of drugs to may be formulated into a slow release formulation (see, for the ocular tissues. Many medications must be applied topi example, Langer (1998) NATURE 392, Supp. 5-10). In some cally to the eye and one common form of treatment is the use of these systems the drug is conjugated with polymers that are of drops or ointments. The topical formulation is adminis 35 degraded, for example, by proteolytic enzymes or by hydroly tered by the patient or caregiver using an eye dropper or sis, to gradually release drug into the target site following dispenser. However, a Substantial disadvantage of this administration. In another approach, drug is trapped through method of drug delivery is that the medication rapidly drains out an insoluble matrix. Following administration, drug then from the ocular surface into the lacrimal system through an is released via diffusion out of, or via erosion of the matrix. opening in the eyelid called the punctum. Furthermore, the 40 Alternatively, drug can be encapsulated within a semi-perme medication is rapidly diluted by the tears secreted by the able membrane or liposome. Following administration, the lacrimal gland. This problem is further compounded by the drug is released either by diffusion through the membrane or patients themselves: one of the principal limitations oftopical via breakdown of the membrane. medication is poor patient compliance. The more often a Specialized deliver systems for the eye, ear, and nasal patient is required to use medication, the less likely they will 45 sinuses have also been developed, and some of these are administer the proper dose at the proper time. described below. Thus, topical treatments do not provide a continuous, pro Ocular Delivery Systems longed delivery of medication and the exact dosage achieved Several systems shown in U.S. patents describe large ocu at the target tissue is unpredictable. Intermittent administra lar inserts to continuously deliver active agents to the eye. tion also is problematic because there is an initial overdosage 50 Certain inserts disperse the drug and require removal of the followed by a rapid decrease in concentration due to dilution carrier of the drug once the drug has been delivered. However, and lacrimal drainage to ineffective levels. U.S. Pat. Nos. 3,845,201, 4,164,559 and 4,179,497 show Another approach for achieving localized drug delivery various inserts in the form of large pellets which dispense involves the injection of drug directly under the conjunctiva drug over a period of time and eventually are completely or tenon's capsule, intra-camerally or intra-vitreally. Unfor 55 eroded, and thus do not require removal after drug delivery. tunately, this approach may require periodic injections of U.S. Pat. No. 4,164,559 describes an ophthalmic drug drug to maintain an effective drug concentration at the target delivery system comprising (a) an enzyme-extracted, chemi site and has many potential adverse effects. cally-modified collagen thin membrane carrier selected from Accordingly, there is a need for a Sustained, controlled, the group consisting of esterified collagen and acylated col delivery system for ophthalmic drugs. 60 lagen and having a pH in the range of 5.5-9.0 whereby the Otic Tissues carrier is soluble in the tear fluid under physiologic condi Similar to ocular liquid treatments, the delivery of thera tions, and (b) an ophthalmically active drug incorporated into peutic agents in a controlled and effective manner to inner ear the carrier. U.S. Pat. No. 4,882,150 describes an ophthalmic tissues is difficult, particularly when considering the tissue drug delivery system, which includes at least one particle of structures of the inner ear (e.g. those portions of the ear 65 bioerodible material, and a liquid or ointment carrier which surrounded by the otic capsule bone and contained within the includes ophthalmic drug to be delivered to the ocular area. temporal bone which is the most dense bone tissue in the The bioerodible material includes collagen. U.S. Pat. No. US 9,289,396 B2 3 4 5.512.301 describes collagen-containing sponges comprising Where the outer layer is impermeable to the drug, it may have an absorbable gelatin sponge, collagen, and an active ingre one or more openings to permit diffusion of the CAI. U.S. dient and the use of the sponges in methods of enhancing Pub. No. 2003/0143280 discloses the use of biodegradable wound healing of external and internal wounds. U.S. Pat. polymer capsules for treating ophthalmic disorder including Nos. 6,197,934 and 6,448,378 describes collagen films which 5 dry eye and glaucoma. The capsules are made of any biode rapidly dissolve at 35°C., methods for preparing the collagen gradable, biocompatible polymer and may contain a treating films, and their use for delivering a dose of therapeutic com agent. pound to a specific tissue site. U.S. Pat. No. 5,418.222 Otic Delivery Systems describes a multi-layered collagen film for use in controlled Otic delivery systems that have been described use natu release of an active ingredient, said film comprising one or 10 rally-occurring materials such as gelatin (e.g., Gelfoam, see, two rate controlling layers and one or more drug reservoir e.g., Silverstein Ann Otol Rhinol Laryngol Suppl. 112:44-8. layers, said layers comprising non-fibrillar collagen and con (1984); Lundman et al. Otolaryngol 1 12:524 (1992); Nedzel tacting each other in a stacked conformation Such that a rate ski et al. Am. J. Otol. 14:278-82 (1993); Silverstein et al. Ear controlling layer is situated at one or both ends of the stack Nose Throat J75:468-88 (1996); Ramsay et al. Otolaryngol. and contacts only one other layer, said other layer being a 15 116:39 (1996); Ruan et al. Hear Res 114:169 (1997); Wana drug reservoir layer. maker et al. Am. J. Otology 19:170 (1998); Arriaga et al. These inserts have certain advantages over liquid treat Laryngoscope 108:1682-5 (1998); and Husmann et al. Hear ments in that a more predictable dosage is obtained because Res 125: 109 (1998)), hyaluronan or hyaluronic acid (see, drug is continuously dispensed over a period of time without e.g., WO 97/38698; Silverstein et al. Am J Otol. 19(2):196 rapid washout. Thus, the unit ocular inserts provide predict 201 (1998)), or fibrin glue or other fibrin-based vehicle (see, able dosage over a period of time without the requirement of e.g., Balough et al. Otolaryngol. Head Neck Surg. 119:427 repeated applications as required with liquid treatments. 31 (1998); Parket al. Laryngoscope 107:1378-81 (1997)). However, the inserts described in these patents are reported to Although these methods may ultimately result in delivery Support drug release only over a period of time ranging from of drug into the inner ear (e.g., by perfusion through the round less than a day to about a week. 25 window membrane), delivery of the therapeutic agent is gen Various ocular drug delivery implants have also been erally not well controlled and/or use of the carrier materials described in an effort to improve and prolong drug delivery. may be associated with adverse side effects. For example, use For example, U.S. Pat. No. 3,949,750 discloses a punctual of gelatin-based materials such as Gelfoam can cause fibrosis plug made of a tissue-tolerable, readily sterilizable material, in the middle ear cavity (see, e.g., Laurent et al. Am. J. such as Teflon, HEMA, hydrophilic polymer, methyl meth 30 Otolaryngol 7(3):181-6 (1986); Liening et al. Otolaryngol. acrylate, silicone, stainless steel or otherinert metal material. Head Neck Surg. 116:454-7 (1997)). Furthermore, naturally The punctual plug may be impregnated with ophthalmic occurring carrier materials generally do not retain their shape medication or may contain a reservoir of the ophthalmic drug. following introduction into the ear (e.g., the materials are U.S. Pat. No. 5,053,030 similarly discloses an intracanalicu naturally viscous or become more liquid upon introduction lar implant. U.S. Pat. No. 5,469,867 discloses a method of 35 into the ear). The changes in the shape of the carrier materials blocking a channel. Such as the lacrimal canaliculus by inject make it extremely difficult to completely retrieve the materi ing a heated flowable polymer into the channel and allowing als from the site of introduction if such should be desired it to cool and solidify. The polymer may be combined with a (e.g., to terminate therapy). It may even prevent delivery of biologically active substance that could leach out of the solid additional therapeutic agents in Subsequent treatments (see, punctum once it has formed in the channel. WO99/37260 40 e.g., Silverstein et al. Am J. Otol 18:586-9 (1997), describing discloses a punctual plug made of a moisture absorbing mate how gelfoam becomes paste-like and prevents future injec rial, which is not soluble in water, such as a modified HEMA. tions of this material from reaching the inner ear fluids). An inflammation inhibitor, Such as heparin, may be added to Sinus (Nasal) Delivery Systems the material from which the punctual plug is made. U.S. Pat. The introduction of drugs directly into the sinuses has been No. 6,196,993 discloses a punctual plug containing glaucoma 45 proposed by others, but has not become a widely used treat medication. The medication is contained in a reservoir within ment technique. For example, U.S. Pub. 2004/01 16958A1 the plug that is in fluid communication with a pore through (Gopferich et al.) describes a tubular sheath or “spacer” which the medication is released onto the eye. WO 2006/ formed of biodegradable or non-biodegradable polymer that, 031658 discloses lacrimal canalicular inserts including a prior to insertion in the patient’s body, is loaded with a con polymer component and a therapeutic component. Similarly, 50 trolled amount of an active Substance. Such as a corticosteroid U.S. Pub. No. 2006/0020248 discloses an ophthalmological or anti-proliferative agent. Surgery is performed to create a device for lacrimal insertion that includes a reservoir for a fenestration in a frontal sinus and the sheath is inserted into medication. U.S. Pub. No. 2004/0013704 discloses Solid or the fenestration. In some embodiments, the sheath is formed semi-solid implant compositions lacking polymeric ingredi of multiple layers of polymeric material, one or more of ents. These implant compositions are made of lipophilic com 55 which is/are loaded with the active substance and one or more pounds and may contain an ophthalmic drug. They may be of which is/are free of the active substance. In other embodi implanted anywhere in the eye including the punctum or ments, the sheath has a “hollow body' which forms a reser lacrimal canaliculous. U.S. Pub. No. 2005/0232972 discloses Voir system wherein the active Substance is contained and a ocular implants to which active agents have been applied to at membrane which controls the release of the active substance least one Surface. In one embodiment, a porous or absorbent 60 from the reservoir. material can be used to make up the entire plug or implant Also, Min, Yang-Gi et al., “Mucociliary Activity and His which can be saturated with the active agent. WO 2004/ topathology of Sinus Mucosa in Experimental Maxilary 066980 discloses a device for delivering a carbonic anhydrase Sinusitis: A Comparison of Systemic Administration of Anti inhibitor (CAI) to the eye over an extended period of time. In biotic and Antibiotic Delivery by Polylactic Acid Polymer.” one embodiment, the device has an inner CAI-containing 65 Laryngoscope, 105:835-842 (August 1995) describes experi core and an outer polymeric layer. The outer layer may be ments wherein experimental sinusitis was induced in three permeable, semi-permeable, or impermeable to the drug. groups of rabbits by pasting the natural sinus ostia, forming US 9,289,396 B2 5 6 an incision and small bore hole made in the anterior wall of ery vehicles and provide an overview of the state of sustained the sinus, introducing pathogenic microbes through the bore drug delivery art. None of these references, however, hole and then closing the incision. Five days after introduc describes specific methods, means, or structures which would tion of the pathogenic microbes, the natural sinus ostia were permit them to be easily adapted for the intended uses tar reopened and the rabbits were divided into three (3) groups. geted in this application. Group 1 (control) received no treatment. Group 2 received In matrix drug delivery devices the drug is dispersed repeated intramuscular injections of ampicillin. In the ani throughout a polymeric matrix and is released as it dissolves mals of Group 3, 1.5 cm x 1.5 cm sheets of polylactic acid or diffuses out of the matrix. Matrix devices have an advan polymer (PLA) film containing amplicillin (0.326 mg/sheet) tage over reservoir devices in that they are not subject to a were rolled up and inserted through the natural ostia into the 10 dose dump if punctured. A disadvantage of matrix devices is infected sinuses. Thereafter, measurements of mucocilliary that it can be difficult to achieve Zero-order drug release transport speed were made and the tissues lining the affected kinetics. Zero-order drug release or near Zero-order drug sinuses were examined histopathologically. The authors con release is desirable because the rate of drug release is inde cluded that the therapeutic effect observed in the animals that pendent of the initial concentration of the drug, thus the drug had received intrasinus implants of PLA/Ampicillin film 15 can be released at therapeutic levels over a sustained period of (Group 3) was significantly better that that observed in the time. The manufacture of matrix devices can also present untreated control animals (Group 1) or those that has received difficulties when the drug and the polymer are processed and repeated intramuscular doses of amplicillin (Group 2). extruded at elevated temperature and/or pressure as this may U.S. Pat. No. 3,948,254 (Zaffaroni), incorporated by ref. reduce the activity of the drug. erence, describes implantable drug delivery devices compris DeVore has previously described several collagen-based ing a drug reservoir Surrounded by a microporous wall. The drug delivery systems, but those systems provided either reservoir may be formed of any of a variety of solid drug rapid release of the therapeutic component or provided an carriers that are permeable to passage of the drug. Zaffaroni initial bolus release of the therapeutic agent followed by a describes a number of applications for the implantable drug time declining release of the therapeutic agent. Such compo delivery devices including placement in a nasal passage. That 25 sitions did not provide desired Zero order release, or involved reference also discusses Zero order release and how Such in vivo polymerizing gels. For example, WO 00/47114 release can be determined. describes an injectable fibrillar collagen Solution comprising Other publications have also reported that introduction of bone morphogenic proteins that polymerizes in situ to form a drugs directly into the paranasal sinuses is effective in the matrix, but in this system 50% of the drug was released within treatment of sinusitis. See, Tarasov, D.I., et al., Vestn Otorino 30 the first 24 hours and the longest period of release measured laringol. Vol. 6, Pages 45-7 (1978). Also, R. Deutschmann, et was 400 hours. WO 00/47130 similarly describes collagen al., Stomat. DDR26 (1976),585-592 describes the placement solutions that convert to gels following in Vivo placement. of a resorbable drug delivery depot within the maxillary sinus The solutions may be used for delivering cells and or drugs to for the purposes of eluting drugs, specifically chlorampheni a tissue by crosslinking them to the collagen using ultraviolet col. In this clinical series a water soluble gelatin was used as 35 radiation. When release of the drug acyclovir was tested in carrier that was mixed with the drug prior to application and this in vivo polymerizing gel system, release was detected at introduced as a mass into the sinus. Since the Substance had the longest time point measured, 28 days. In an abstract little mechanical integrity and dissolved in a relatively short (DeVore et al., Abstract 5126-B524, ARVO, 2006), propose timeframe, to achieve a therapeutic effect, the author Sug the use of this system to deliver sustained release of growth gested that it must be instilled every 2 to 3 days. U.S. Pat. No. 40 factors and other agents for treatment of retinal disorders for 6,398.758 to Jacobsen et al. describes a hollow cylindrical up to 2 months, but no details were given. In ARVO abstract sponge loaded with drug and pressed against a blood vessel 480-B454 (Invest Ophthalmol Vis Sci 2005; 46:) release and wall. This allows the drug to contact the wall while sustaining diffusion of dexamethasone from collagen gels and films blood flow within the center of the lumen. Further, a skin is through human Scleral tissue was measured for a 24 hour provided to direct the drug into the walls of the blood vessel 45 period. No long-term release was evaluated. In ARVO and prevent drug from flowing into the lumen. While sponges abstract 1816-D686 (Invest Ophthalmol Vis Sci 2008; 49: loaded with drug at the time of their application do permit E-Abstract 1816) release of POT-4 (a strong complement Some degree of Sustained release, the time required to load inhibitor peptide) from collagen gel was bi-phasic with an them also correlates closely with the time over which they initial burst followed by sustained release. will elute substance. Thus, if delivery is required for a longer 50 The collagen-based drug delivery constructs of the present period of time additional mechanisms must be employed to invention address deficiencies in the art by providing matrices regulate their release. that achieve Zero-order or near Zero-order drug-release kinet Many of the devices described above are reservoir-type ics typically associated with reservoir devices, but without the drug-delivery devices that containa receptacle or chamber for risk of dose dumping and the manufacturing difficulties of storing the drug. There are drawbacks to reservoir drug deliv 55 reservoir devices. Further, while the collagen-based drug ery devices in that they are difficult to manufacture, difficult delivery constructs are illustrated in the context of oph to achieve drug content uniformity (i.e., device to device thalmic, otic, and sinus drug delivery, those uses are illustra reproducibility, particularly with small ocular devices), and tive only as the collagen-based drug delivery constructs can they carry the risk of a "dose dump” if they are punctured. be used anywhere in the body that prolonged delivery of a There are also several examples in the patent literature 60 precise dose of drug or other agent is needed. where various Sustained release mechanisms have generally been proposed using systems with pre-incorporated drugs SUMMARY OF INVENTION into matrices or polymers. These include U.S. Pat. No. 3,948, 254 (Zafferoni), US 2003/0185872A2 (Kochinke), WO The present application provides collagen-based con 92/15286 (Shikani), and U.S. Pat. No. 5,512,055 (Domb, et 65 structs, such as film, wafer, or membrane-type delivery sys al.). In general, these references discuss various materials and tems, for use in Sustained and/or controlled release of an agent structures that may be used to construct Sustained drug deliv or drug. In one aspect, the film, wafer, or membrane is com US 9,289,396 B2 7 8 prised of at least one layer composed of crosslinked, chemi thalmic, otic, and sinus (nasal) drugs. Examples of such drugs cally derivatized collagen. The collagen may be chemically are given in the Detailed Description. In some embodiments, derivatized to change the net charge on collagen to enhance the drugs or agents are those that have low water or aqueous ionic binding of active agents or drugs and wherein said film solubility. Thus, in particular aspects, the collagen-based con or membrane delivery systems may be multi-layer in compo- 5 structs comprise an ophthalmic, otic, or sinus (nasal) drug sition where at least one layer is a rate controlling layer that has low water or aqueous liquid solubility. composed of derivatized collagen on one Surface of the film or One example of a drug with low water solubility that may membrane providing a barrier to diffusion of the active agent be delivered using the collagen-based constructs of the inven or drug. tion is Latanoprost, which is commonly used in methods of In one embodiment, the collagen in the delivery construct 10 is acylated to alter ionic charge and charge density and to treating glaucoma. In some embodiments, the collagen con provide a soluble collagen composition at neutral pH. In struct delivers an extended therapeutic concentration of another embodiment, the collagen is altered to increase net Latanoprost that is at least about 150 ug per film, wafer, or negative charge by reacting the collagen solution with acy membrane to provide an effective dosage of about 1.5ug per lating agents selected from the group consisting of anhy- 15 day for at least about 90 days. In other embodiments, the drides including maleic anhydride. Succinic anhydride, glu extended therapeutic concentration of Latanoprost is at least taric anhydride, citractonic anhydride, methyl Succinic about 300 ug per film, wafer, or membrane to provide an anhydride, itaconic anhydride, methyl glutaric anhydride, effective dosage of about 1.5ug per day for at least about 180 dimethyl glutaric anhydride, phthalic anhydride, and many days. In some embodiments, the delivery is Sustained, Zero other Such anhydrides; acid chlorides including oxalyl chlo- 20 order delivery. In other embodiments, the delivery includes a ride, malonyl chloride; sulfonyl chlorides including chloro bolus release at about day 1-3, followed by controlled deliv sulfonylacetyl chloride, chlorosulfonylbenzoic acid, ery of a therapeutically effective amount of Latanoprost for at 4-chloro-3-(chlorosulfonyl)-5-nitrobenzoic acid, 3-(chloro least about 6 months. In some embodiments, the delivery is Sulfonyl)-P-anisic acid, and others; Sulfonic acids including for at least about 6 months but not for more than one year. 3-sulfobenzoic acid and others. 25 Other delivery profiles and time periods are set forth in the In some embodiments, the concentration of acylating agent Detailed Description and Examples. to chemically derivatize collagen is less than 5% (weight per In certain aspects, the delivery system comprises a col weight collagen Solids) to minimally increase net negative lagen-based film, wafer, or membrane as described above that charge. Alternatively, the concentration of acylating agent to further comprises a layer which is a barrier to drug diffusion. chemically derivatize collagen is less than 20% (weight per 30 In these embodiments, the films, wafers, or membranes pro weight collagen solids) to moderately increase net negative vide essentially unidirectional delivery of the selected drug charge. In still other embodiments, the concentration of acy through the non-barrier layer or layers. In some embodi lating agent to chemically derivatize collagen is more than ments, the barrier layer is composed of collagen acylated with 25% (weight per weight collagen Solids) to maximally chemical agents imparting chemical barriers to diffusion of increase net negative charge. In one embodiment, the col- 35 selected drugs. Examples of acylating agents that may be lagen is altered to increase net positive charge by reacting the used to produce the barrier layer are those agents producing collagen solution with acylating agents selected from 4.6- biologically compatible collagenous reaction products com diamino-2-methylthiopyrimidine-5-sulfonic acid. In still prising ethylenically unsaturated monomerically substituted other embodiments, the concentration of acylating agent to collagen, the monomeric Substituents being essentially free chemically derivatize collagen is less than 5% (weight per 40 of nitrogen, e.g., methyacrylate, styrene, polyvinyl, ethylene. weight collagen Solids) to minimally increase net negative In still another aspect, the invention provides a method for charge. And in yet other embodiments, the concentration of preparing a collagen-based, film or construct comprising a acylating agent to chemically derivatize collagen is less than therapeutic drug and capable of providing Sustained and/or 20% (weight per weight collagen solids) to moderately controlled release of that drug, the method comprising: increase net negative charge. In still other embodiments, the 45 (i) preparing derivatized collagen; concentration of acylating agent to chemically derivatize col (ii) dissolving the derivatized collagen precipitates in lagen is more than 25% (weight per weight collagen Solids) to physiological buffer at concentrations ranging from 10 maximally increase net negative charge. mg/mL to 100 mg/mL, In yet another aspect, the invention provides a delivery (iii) adding active drug in a concentration that provides system or collagen-based construct in which films, wafers, or 50 sustained release at therapeutic levels for at least 3 to 6 membranes of acylated collagen containing extended thera months; peutic concentrations of selected drugs are exposed to ultra (iv) optionally centrifuging the drug-containing, deriva violet irradiation in an oxygen-free (for example, a nitrogen) tized collagen to remove air bubbles; atmosphere for time periods ranging from about 2 minutes to (V) casting the collagen-drug solution into a thin layer or about 20 minutes. In some embodiments the exposure is for 55 onto a mold; time periods ranging from about 14 to about 19 minutes, (vi) drying or partially drying the solution to form a film, about 15 to about 19 minute, about 16 to about 18 minutes, membrane, or wafer; about 15, about 16, about 17, about 18, or about 19 minutes. (vii) exposing the film, membrane, or wafer to ultraviolet In Some embodiments, the exposure time is approximately 18 (“UV) radiation in an oxygen-free atmosphere, such as minutes. Thus, in Some embodiments, the delivery system is 60 a nitrogen atmosphere, for from about 17 to about 19 exposed to ultraviolet irradiation in a nitrogen atmosphere to minutes; and produce collagen films, wafers, or membranes with defined (viii) recovering the UV-crosslinked, drug-containing, resorption characteristics such that the film, wafer, or mem film, membrane, or wafer. brane delivers therapeutically effective amounts of drug for a In some embodiments, the concentration of the derivatized period of time of at least about 3 to 6 months. 65 collagen in step (ii) is adjusted to a range between 15 and 100 In one aspect, the collagen constructs of the invention are mg/mL, between about 25 and about 60 mg/mL, or between used to deliver extended therapeutic concentrations of oph about 30 and about 45 mg/mL. US 9,289,396 B2 10 In some embodiments, the ultraviolet radiation exposure in provide an effective dosage of about 1.5ug per day for at least step (vii) is for about 18 minutes. about 180 days of sustained and/or controlled delivery. In some embodiments, the UV-crosslinked collagen con In this aspect as well, the collagen-based film, wafer, or struct is further treated by incubating it in a solution, for membrane of may be one in which the Latanoprost present in example a saline solution. In some embodiments, the Solution 5 the film, wafer, or membrane is resistant to removal by a 1 day treatment is for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 treatment in an aqueous buffer. Likewise, the collagen-based days, 7, days, about 1 week, about 2 weeks, or about 3 weeks. film, wafer, or membrane may be one in which the Latano In some embodiments the Solution treatment results in col prost present in the film, wafer, or membrane is resistant to lagen construct that releases drug with Zero order kinetics or removal by a 7 day treatment in an aqueous buffer. with near Zero order kinetics. In still other embodiments, the 10 Solution treatment results in collagen-based construct that In this aspect as well as any other aspects of the invention, releases drug with an initial bolus until about day 1, day 2, or the film, wafer, or membrane may further comprise a barrier day 3, followed by controlled release for at least about 3, 4, 5, layer of collagen derivatized by an acylation agent. 6, 7, 8, 9, 10, 11, or 12 months. In still further embodiments, In yet another aspect, the invention provides a method for the controlled release is for at least 6 months, but not for more 15 preparing a collagen-based film, wafer, or membrane con than about 7, 8, 9, 10, 11, or 12 months. struct comprising atherapeutic drug and capable of providing In some embodiments, the method includes partial drying sustained and/or controlled release of that drug, the method of the Solution and an additional step of applying a barrier comprising: layer comprising collagen derivatized by an acylation agent (i) preparing derivatized collagen; that imparts a barrier to diffusion of the drug to one side of the (ii) dissolving the derivatized collagen precipitates in drug reservoir layer prior to ultraviolet radiation of the film or physiological buffer at concentrations ranging from 10 membrane. mg/mL to 100 mg/mL, Collagen-based film, wafers, or membrane prepared by the (iii) adding active drug in an amount and concentration methods are of course part of the invention. sufficient to provide sustained release at therapeutic lev In one particular aspect, the invention provides a collagen 25 els for at least 3 to 6 months; based film, wafer, or membrane delivery system for use in (iv) optionally centrifuging the drug-containing, deriva Sustained and/or controlled release of an extended therapeutic tized collagen to remove air bubbles; concentration of Latanoprost, said film or membrane com (V) casting the collagen-drug solution into a thin layer or prising at least about 150 ug of Latanoprost in one or more onto a mold; layers of glutaric anhydride-derivatized collagen, wherein the 30 (vi) drying or partially drying the solution to form a film, film or membrane has been crosslinked with ultraviolet radia membrane, or wafer; tion in a nitrogen atmosphere for from 17 to 19 minutes. In (vii) exposing the film, membrane, or wafer to ultraviolet one specific embodiment, the film, wafer, or membrane pro (“UV) radiation in a low oxygen atmosphere for from vides controlled release of about 1.5 ug Latanoprost per day about 17 to about 19 minutes; and for at least about 90 days. In certain embodiments, therefore, 35 (viii) recovering the UV-crosslinked, drug-containing, the film, wafer, or membrane may be used in a method of film, membrane, or wafer. treating glaucoma comprising administering to the eye of a In one embodiment, the ultraviolet radiation exposure in glaucoma patient the collagen-based film, wafer, or mem step (vii) is for about 18 minutes. In other embodiments, the brane delivery system comprising Latanoprost. method further comprises treating the UV-crosslinked col In an alternate aspect, the invention provides a collagen 40 lagen construct an aqueous solution for about 1, 2, 3, 4, 5, 6, based, ultraviolet radiation crosslinked, film, wafer, or mem or 7 days. In certain embodiments, the drug is Latanoprost in brane construct comprising at least one layer of chemically an amount Sufficient to release an effective dosage of about derivatized collagen and an amount of an ophthalmic, otic, or 1.5 ug per day for at least about 180 days. In still other nasal drug that has low water or aqueous solubility, wherein embodiments, the method employs partial drying of the col the amount of the drug is Sufficient to provide a therapeuti 45 lagen-drug solution in step (vi) and wherein the method fur cally effective amount of drug per day over a period of about ther comprises applying a barrier layer comprising collagen six months, and further wherein the construct has been derivatized by an acylation agent that imparts a barrier to exposed to ultraviolet radiation crosslinking for a period of 17 diffusion of the drug to one side of the film, wafer, or mem to 19 minutes in a low oxygen environment. In one embodi brane prior to ultraviolet radiation step (vii). ment, the drug present in the film, wafer, or membrane is 50 In this aspect as well, the invention also encompasses any resistant to removal by a 1 day treatment in an aqueous buffer, collagen-based film or membrane prepared by the method while in another embodiment it is resistant to removal by a 7 described above. day treatment in an aqueous buffer. In other aspects the invention includes methods of treating In certain embodiments, the film, wafer, or membrane fur ophthalmic, otic, and sinus diseases by administering a col ther comprises a barrier layer of collagen derivatized by an 55 lagen-based construct of the invention containing an oph acylation agent. thalmic, otic, or sinus drug, respectively. Accordingly, the In still another aspect, the invention provides a collagen invention also encompasses the use of any of the collagen based, ultraviolet radiation crosslinked, film, wafer, or mem based constructs in methods of treating. In one particular brane delivery system comprising at least one layer of glutaric aspect, a collagen-based construct comprising Latanoprost is anhydride-derivatized collagen and at least about 150 g of 60 used in a method of treating glaucoma comprising adminis Latanoprost, wherein the film, wafer, or membrane has been tering to the eye of a glaucoma patient a collagen-based, crosslinked with ultraviolet radiation for from 17 to 19 min ultraviolet radiation crosslinked, film, wafer, or membrane utes. In some embodiments, the film, wafer, or membrane delivery system comprising Latanoprost as described herein. provides Sustained and/or controlled release of about 1.5 ug It is to be understood that both the foregoing general Latanoprost per day for at least about 6 months but for less 65 description and the following detailed description are exem than one year. In other embodiments, the film, wafer, or plary and explanatory only and are not restrictive of the inven membrane comprises at least about 300 ug Latanoprost to tion as claimed. US 9,289,396 B2 11 12 BRIEF DESCRIPTION OF THE DRAWINGS diffusion of hydrophilic drugs or agents and it may be hydro philic surface to retard or prevent diffusion of hydrophobic FIG. 1 depicts the release of Latanoprost from in situ agents or drugs. polymerizing collagen gels, as measured by HPLC. Diffusion flux of therapeutic agents is dependent on ionic FIG. 2 depicts the release of Latanoprost from collagen interactions between the collagen composition and the thera wafers/films as measured by EIA. peutic agent, concentration of collagen composition and FIG. 3 depicts the release of Latanoprost from collagen therapeutic agent, the aqueous solubility of the therapeutic wafers/films pre-treated in buffer. agent, and diffusion barrier effectiveness. As described in more detail below, in some embodiments, the ionic properties DETAILED DESCRIPTION OF THE INVENTION 10 of the collagen base may be altered by derivatizing collagen with agents that alter net charge to make the collagen more The present application describes methods of preparing anionic or more cationic. degradable collagen-based constructs that include therapeu In some embodiments, the resorption rate of the collagen tic drugs or compounds and methods of using those con films and membranes is controlled by the degree of crosslink structs to deliver drug to a particular tissue or site, including 15 ing accomplished by controlling the time of ultraviolet optic, otic, and sinus tissues. (“UV) irradiation. In one embodiment, the collagen-based constructs com In some embodiments, the collagen-based construct pro prise a single drug reservoir alone with no rate controlling vides Sustained release of a drug or other agent. “Sustained barrier. In another embodiment, the collagen-based con release' means release of a drug or other agent from a col structs comprise at least one rate controlling barrier layer, said lagen-based film, membrane, wafer, or other construct for a layers co-bonded to form a film construct such that the rate period of time that is greater than one month. In some controlling barrier is located on one side of the construct to embodiments, the release is for greater than two months. In provide a barrier to drug diffusion in one direction and permit other embodiments, the release is for greater than about 3 unidirectional delivery of said drug. In some examples, the months, 4 months, 5 months, 6 months, 7 months, 8 months, drug reservoir layer and/or the rate controlling barrier layers 25 9 months, 10 months, 11 months, or even 12 months. In some have a thickness of from about 0.01 to about 1 mm, often from embodiments, the release is for greater than 6 months but for about 0.05 to about 0.5 mm, and in some cases from about less than one year, for greater than 6 months but for less than 0.02 to about 0.2 mm. 9 months; for greater than 6 months but for less than 8 months; The collagen-based constructs are designed to provide Sus or for greater than 6 months but for less than 7 months. tained release of drugs, including long-term and long-term 30 In one embodiment, the collagen-based construct provides release with Zero order, or near Zero order release kinetics. In controlled release of a drug or other agent. “Controlled some embodiments, the base collagen composition can be release” means release of a drug or agent from a collagen chemically derivatized to alter the overall ionic characteris based film, membrane, wafer or other construct described tics to better bind the drug component thereby altering the rate herein that follows Zero order, or nearly Zero order, release for diffusion into tissues. Such constructs can be implanted to 35 greater than one month, greater than 2 months, greater than provide Sustained and controlled release of therapeutic agents about 3 months, about 4 months, 5 months, 6 months, 7 for ocular, otic, or sinus applications. months, 8 months, 9 months, 10 months, 11 months, or even The implant site varies depending upon the indication. In 12 months. In some embodiments, the release is for greater Some embodiments the implant site is Submucosally. In other than 6 months but for less than one year; for greater than 6 embodiments, the implant is transcleral, under a Sclera flap, 40 months but for less than 9 months; for greater than 6 months adjacent to the Sclera, in the Subjacent Sclera near the macula, but for less than 8 months; or for greater than 6 months but for episcleral, Subconjunctival, Sub tenon, or in a fistula or fen less than 7 months. In some embodiments, controlled release estration, such as a fistula or fenestration created by Surgery. includes an initial bolus release on day 1 or on about days 1 to Examples of implant sites are also provided in the Examples 3. In other embodiments, the controlled release does not and throughout the Summary of the Invention and Detailed 45 include a bolus release on day 1 so that Zero order, or nearly Description. In some embodiments, combinations of the sites Zero order, release begins on day 1. In still other embodi are used. ments, the controlled release does not include a bolus release Depending upon the embodiment, the constructs can be in after day 3, so that Zero order, or nearly Zero order, release the form of single layer films, wafers, or membranes, multi begins on about day 4. A controlled release is a Sustained layer films, wafers, or membranes, unidirectional single layer 50 release, but a Sustained release is not necessarily a controlled films, wafers, or membranes, or unidirectional multilayer release. films, wafer, or membranes. In some embodiments, the film, In some embodiments, the collagen-based constructs pro wafer, or membrane constructs are composed of collagen that vide Sustained release and/or controlled release of a drug or is chemically derivatized to alter the ionic properties to more other agent that is sparingly soluble in aqueous solution but efficiently bind specific agents or drugs to improve Sustained 55 soluble in alcohols and other water miscible solvents mixed delivery. Therapeutic agents or drugs are added to the films into the collagen composition. Examples of Such drugs and and membranes in quantities to provide uniform Sustained, agents are provided below and in the Examples. and in some embodiments, controlled, delivery for various In one embodiment, the disclosure provides a method for therapeutic time intervals. In those embodiments involving preparing a collagen-based film, membrane, wafer or con unidirectional films or membranes, a physical or chemical 60 struct for Sustained release of a therapeutic drug, comprising: barrier is applied to prevent diffusion of drugs and agents in (i) preparing derivatized collagen; one direction. In some embodiments, physical barriers are (ii) dissolving the derivatized collagen precipitates in used in the form of an occlusive film bound to the base physiological buffer at concentrations ranging from 10 collagen construct. In other embodiments, chemical barriers mg/mL to 100 mg/mL, are produced by chemically derivatizing one Surface of the 65 (iii) adding active drug in a concentration that provides collagen constructs to create an occlusive barrier. The chemi sustained release at therapeutic levels for at least 3 to 6 cal barrier may be a hydrophobic surface to retard or prevent months; US 9,289,396 B2 13 14 (iv) optionally centrifuging the drug-containing, deriva in pyrogen-free deionized water, and then redissolved in tized collagen to remove air bubbles: phosphate buffer (0.01 M phosphate buffer, pH 7.4) to (V) casting the collagen-drug solution into a thin layer or achieve a final concentration of approximately 30 mg/ml. onto a mold; A barrier layer is prepared by derivatized purified collagen (vi) drying or partially drying the solution to form a film, with glutaric anhydride and B-styrene Sulfonyl chloride as membrane, or wafer; previously described (U.S. Pat. No. 5,480,427). In general, (vii) exposing the film, membrane, or wafer to ultraviolet the collagen solution is adjusted to pH 9.0 with 10 N and 1 N (“UV) radiation in an oxygen-free atmosphere, such as NaOH. While stirring the solution, glutaric anhydride is a nitrogen atmosphere, for from about 17 to about 19 added at 10% (weight of collagen). For 5 minutes, the stirring minutes; and 10 (viii) recovering the UV-crosslinked, drug-containing, is continued, and the pH maintained and 2% B-styrene Sulfo film, membrane, or wafer. nyl chloride added at pH 9.0 and stirred. The pH of the In some embodiments, the concentration of the derivatized solution is then adjusted to 4.3 to precipitate the derivatized collagen in step (ii) is adjusted to a range between 15 mg/mL collagen. The precipitate is centrifuged, washed one time, and and 100 mg/mL, in other embodiments the concentration is 15 then redissolved in phosphate buffer (0.01 M phosphate adjusted to between about 25 mg/mL and about 60 mg/mL, buffer, pH 7.4) to achieve a final concentration of approxi while in still other embodiments, the concentration is mately 30 mg/ml. adjusted to between about 30 mg/mL and about 45 mg/mL. Uni-directional collagen films containing drug or agent is In some embodiments, the ultraviolet radiation exposure in prepared by mixing the appropriate concentration of the drug step (vii) is for about 18 minutes. or agent with glutaric anhydride-derivatized collagen (reser In some embodiments, the UV-crosslinked film, mem voir layer) described above. The mixture was centrifuged at brane, or wafer is further treated by incubating it in a solution, 3500 rpm for 10 minutes if air bubbles are observed. The for example a saline Solution. In some embodiments, the mixture is then poured into a mold and partially dried until it Solution treatment is for about 1 day, 2 days, 3 days, 4 days, 5 appears slightly gelatinous. A layer of barrier film collagen days, 6 days, 7 days, about 1 week, about 2 weeks, or about 25 composition is the carefully placed on the surface of the 3 weeks. The solution treatment helps to eliminate or reduce reservoir layer and the molds placed again in the sterile lami any initial bolus release of drug. For example, in some nar flow hood to fully dehydrate the unidirectional film. embodiments the Solution treatment results in a film, mem Release kinetics are adjusted as described for the non-unidi brane, or wafer that releases drug with Zero order kinetics. In rectional constructs. Films with a barrier layer are particularly other embodiments, the solution treatment results in a film, 30 useful for applications to the back of the eye so that the drug membrane, or wafer that releases drug with near Zero order is effectively delivered in therapeutic levels to the target struc kinetics. In still other embodiments, the solution treatment tures in the eye and not distributed to all surrounding tissues. results in a film, membrane, or wafer that releases drug with The barrier containing films of this invention relate to col an initial bolus until about day 1, day 2, or day 3, that is then lagen-based constructs or devices and methods for local drug followed by controlled release for at least about 3, 4, 5, 6, 7, 35 delivery, and in particular directed to an implantable system 8, 9, 10, 11, or 12 months. In still further embodiments, the intended to deliver therapeutic agents to a target tissue while controlled release is for at least 6 months, but not for more limiting exposure to Surrounding tissues and fluids while still than about 7, 8, 9, 10, 11, or 12 months. achieving Sustained drug levels, regionally or systemically. In another embodiment, the method comprises preparing Adjusting Ionic Characteristics of Base Collagen Composi unidirectional collagen-based film constructs. In these 40 tions embodiments, the drug reservoir layer is prepared generally Acylation reactions to derivatize soluble and insoluble col as described above. However, following casting the solution lagen have been described by DeVore, et al. in a series of into a thin layer and partial dehydration, a second barrier layer patents (U.S. Pat. Nos. 4.713,446, 4,851,513, 4,969,912, is applied to one side of the drug reservoir layer. The collagen 5,067,961, 5,104,957, 5,201,764, 5,219,895, 5,332,809, composition for this layer is prepared by derivatizing col 45 5,354,336, 5,476,515, 5,480,427, 5,631,243, and 6,161,544, lagen with an acylation agent that imparts a barrier to diffu each incorporated by reference). An increase in net negative sion of the active agent. If the active agent is hydrophilic, the charge density will increase ionic binding of agents with a net barrier layer should be hydrophobic. For example, soluble positive charge. collagen can be derivatized with agents such as B-styrene Specific acylation agents have been used to alter the net sulfonyl chloride or polyvinyl sulfonic acid, ethylene/maleic 50 charge and charge density of intact tissue proteins. Certain anhydride copolymer or combinations of glutaric anhydride/ agents can be used to change the net charge from positive to B-styrene Sulfonyl chloride, glutaric anhydride/polyvinyl Sul negative. These agents include, but are not limited to, anhy fonic acid, glutaric anhydride/ethylene/maleic anhydride drides including maleic anhydride. Succinic anhydride, glu copolymer or other combinations of anhydrides, acid chlo taric anhydride, citractonic anhydride, methyl succinic anhy rides, Sulfonyl chlorides, Sulfonic acids producing hydro 55 dride, itaconic anhydride, methyl glutaric anhydride, philic derivatized collagen with anhydrides, acid chlorides, dimethyl glutaric anhydride, phthalic anhydride, and many Sulfonyl chlorides, or Sulfonic acids producing hydrophobic other such anhydrides. Acid chlorides include, but are not derivatized collagens. limited to, oxalyl chloride, malonyl chloride, and many oth To prepare a unidirectional film, wafer, or membrane, a ers. Sulfonyl chlorides include, but are not limited to, chlo reservoir layer is prepared using purified, pepsin digested 60 rosulfonylacetyl chloride, chlorosulfonylbenzoic acid, collagen derivatized with glutaric anhydride as previously 4-chloro-3-(chlorosulfonyl)-5-nitroebnzoic acid, 3-(chloro described (U.S. Pat. Nos. 5,631,243 and 5.492,135). In gen sulfonyl)-P-anisic acid, and others. Sulfonic acid include, but eral, the collagen solution is adjusted to pH 9.0 with 10 N and are not limited to, 3-sulfoebnzoic acid and others. 1 N NaOH. While stirring the solution, glutaric anhydride is Certain agents can change the net charge from one positive added at 10% (weight of collagen). The pH of the solution is 65 to two negatives per reacted site. Specific agents include, but then adjusted to 4.3 with 6 N and 1 NHCl to precipitate the are not limited to, 3,5-dicarboxybenzenesulfonyl chloride derivatized collagen. The precipitate is centrifuged, washed and others. US 9,289,396 B2 15 16 Certain agents can be used to change the net charge from A discussion of low water and aqueous solubility drugs for positive to neutral per reacted site. Specific agents include, ophthalmic applicationis presented in US 2010/09008993 A1 but are not limited to, anhydrides including acetic anhydride, (Proksch & Ward). chloroacetic anhydride, propionic anhydride, butyric anhy Examples of ophthalmic drugs with low water or aqueous dride, isobutyric anhydride, isovaleric anhydride, hexanoic liquid solubility included in the compositions of the present anhydride, and other anhydrides; acid chlorides including invention are selected from the group consisting of anti-in acetyl chloride, propionyl chloride, dichloropropionyl chlo flammatory agents, anti-infective agents (including antibac ride, butyryl chloride, isobutyryl chloride, Valeryl chloride, terial, antifungal, antiviral, antiprotozoal agents), anti-aller and others; Sulfonyl chlorides including, but not limited to, gic agents, antihistamines, antiproliferative agents, anti 10 angiogenic agents, anti-oxidants, antihypertensive agents, ethane sulfonyl chloride, methane sulfonyl chloride, 1-butane neuroprotective agents, cell receptor agonists, cell receptor sulfonyl chloride, and others. antagonists, immunomodulating agents, immunosuppressive Certain agents can be used to change the net charge from agents, intraocular (“IOP) lowering agents, carbonic anhy one positive to two positives per reacted site. Specific agents drase inhibitors, cholinesterase inhibitor miotics, prostaglan include, but are not limited to, 4,6-diamino-2-methylthiopy 15 dins and prostaglandin receptor agonists, prostaglandin F rimidine-5-Sulfonic acid, and others. derivatives, prostaglandin F receptor antagonists, Stability of Collagen Constructs cyclooxygenase-2 inhibitors, muscarinic agents, and combi While the stability and durability of the different collagen nations thereof. drug delivery constructs can be assessed by monitoring drug Some non-limiting examples of ophthalmic drugs and their release, other tests may also be used to measure their stability. corresponding solubility in water are given in Table 1: For example, the robustness of the collagen constructs can be tested with trypsin or collagenase. For the trypsin digestion TABLE 1 test, a collagen film, wafer, membrane, etc. is evaluated by incubating it without drug in 0.02% trypsin solution mixed in Solubility of Ophthalmic Drugs 1M Trizma buffer, pH 8.0 containing sodium chloride, cal 25 Drug Name Approx. Solubility in water (mg/mL) cium chloride and magnesium chloride. After 24 hours of incubation at 37° C. vials are centrifuged to recover the NSAIDS Supernatant and analyzed for hydroxyproline using standard indomethacin O.O1 assays. The degradation profile is determined by calculating piroxicam O.O3 the hydroxyproline present in the incubation Supernatant, 30 ketoprofen O.11 nepafenac O.O2 which is indicative of collagen degraded as a result of trypsin flurbiprofen O.OO8 digestion. A stable collagen construct should show negligible diclofenac O.OOO8 when incubated in trypsin. etodolac O.O16 For the collagenase test, a collagen film, wafer, membrane, Suprofen O.04 35 naproxen O.O16 etc. is incubated without drug in bacterial collagenase in bromfenac O.O13 sodium phosphate buffer, pH 7.4. After 24 hours of incuba Immunosupressive Agents tion at 37°C., vials are centrifuged to recover the supernatant and analyzed for hydroxyproline using standard assays. The cyclosporin O.OO95 degradation profile is determined by calculating the hydrox Steroids 40 yproline present in the incubation Supernatant, which is dexamethasone O.OO9 indicative of collagen degraded as a result of collagenase betamethasone O.OOOS digestion. The degree of degradation is shown to be depen triamcinolone O.O8 dent on collagenase concentration. loteprednoletabonate O.OO7 Exemplary Drugs for Delivery Antiglaucoma A variety of drugs are suitable for incorporation in the 45 epinephrine O.18 collagen constructs, consistent with their known dosages and dipivefrin O.OS8 demecarium O.OOO1 uses. Thus, although certain examples are mentioned below latanoprost O.OS2 and in the Examples section, those drugs are exemplary only. bimatoprost O.O2 The inventors recognize that the collagen constructs provide travoprost O.OO8 a generalizable drug delivery system that can be used with 50 Antihistamine many different therapeutic, or even diagnostic, agents or drugs. ketotifen O.OO8 As shown in the Examples, the collagen constructs described herein, including pretreated collagen constructs, Non-limiting examples of the glucocorticosteroids are: provide Sustained and Zero order, or near Zero order release of 55 21-acetoxypregnenolone, alclometaSone, algestone, amcino the drug Latanoprost (13,14-dihydro-17-phenyl-18, 19.20 nide, beclomethasone, betamethasone, budesonide, chloro trinor-prostaglandin F2a-1-isopropyl ester). Latanoprost prednisone, clobetasol, clobetaSone, clocortolone, clopred exhibits limited aqueous solubility (about 50 ug/mL in phos nol, corticosterone, cortisone, cortivaZol, deflazacort, phate buffer) and is supplied in methyl acetate. It is readily desonide, desoximetaSone, dexamethasone, diflorasone, miscible in ethanol and can be mixed in with soluble collagen. 60 diflucortolone, difluprednate, enoxolone, fluazacort, fluclo Thus, the results obtained with Latanoprost provide a specific ronide, flumethasone, flunisolide, fluocinolone acetonide, example that provides evidence that other drugs and agents fluocinonide, fluocortin butyl, fluocortolone, fluo with low water or aqueous liquid solubility can also be incor rometholone, fluperolone acetate, fiuprednidene acetate, flu porated into the collagen film/wafers described herein to pro prednisolone, flurandrenolide, fluticaSone propionate, for vide delivery that is sustained (for example, 1, 2, 3, 4, 5, or 6 65 mocortal, halcinonide, halobetasol propionate, months or more) and/or controlled with either Zero order or halometaSone, halopredone acetate, hydrocortarnate, hydro near Zero order release. cortisone, loteprednol etabonate, maZipredone, medrysone, US 9,289,396 B2 17 meprednisone, methylprednisolone, mometaSone furoate, ran, cefpimizole, cefpiramide, cefpirome, cefpodoxime proX paramethasone, prednicarbate, prednisolone, prednisolone etil, cefprozil, cefroxadine, cefsulodin, ceftazidime, 25-diethylamino-acetate, prednisolone sodium phosphate, cefiteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, prednisone, prednival, prednylidene, rimexolone, tiXocortol, cefuroxime, cefuZonam, cephacetrile sodium, cephalexin, triamcinolone, triamcinolone acetonide, triamcinolone bene cephaloglycin, cephaloridine, cephalosporin, cephalothin, tonide, triamcinolone hexacetonide, their physiologically cephapirin Sodium, cephradine, piveefalexin), cephamycins acceptable salts, derivatives thereof, combinations thereof, (e.g., cefbuperaZone, cefinetazole, cefininox, cefotetan, and mixtures thereof. In one embodiment, the therapeutic cefoxitin), monobactams (e.g., aztreonam, carumonam, tige agent is selected from the group consisting of difluprednate, monam), oxacephems, flomoxef, moxalactam), penicillins loteprednol etabonate, prednisolone, combinations thereof, 10 (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, ampi and mixtures thereof. cillin, apalcillin, aspoxicillin, azidocillin, azlocillin, Non-limiting examples of the non-steroidal anti-inflam bacampicillin, benzylpenicillinic acid, benzylpenicillin matory drugs ("NSAIDs) are: aminoarylcarboxylic acid Sodium, carbenicillin, carindacillin, clometocillin, cloxacil derivatives (e.g., enfenamic acid, etofenamate, flufenamic lin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacil acid, isonixin, meclofenamic acid, mefenamic acid, niflumic 15 lin, hetacillin, lenampicillin, metampicillin, methicillin acid, talniflumate, terofenamate, tolfenamic acid), arylacetic Sodium, mezlocillin, nafcillin Sodium, oxacillin, penamecil acid derivatives (e.g., aceclofenac, acemetacin, alclofenac, lin, penethamate hydriodide, penicillin G benethamine, peni amfenac, amtolimetin guacil, bromfenac, bufexamac, cin cillin G benzathine, penicillin G benzhydrylamine, penicillin metacin, clopirac, diclofenac sodium, etodolac, felbinac, fen G calcium, penicillin Ghydrabamine, penicillin G potassium, clozic acid, fentiazac, glucametacin, ibufenac, indomethacin, penicillin G procaine, penicillinN, penicillin O, penicillin V. isofeZolac, isoxepac, lonazolac, metiazinic acid, mofeZolac, penicillin V benzathine, penicillin V hydrabamine, penime Oxametacine, piraZolac, proglumetacin, Sulindac, tiaramide, picycline, phenethicillin potassium, piperacillin, pivampicil tolimetin, tropesin, Zomepirac), arylbutyric acid derivatives lin, propicillin, quinacillin, Sulbenicillin, Sultamicillin, (e.g., bumadizon, butibufen, fenbufen, Xenbucin), arylcar talampicillin, temocillin, ticarcillin), lincosamides (e.g., clin boxylic acids (e.g., clidanac, ketorolac, tinoridine), arylpro 25 damycin, lincomycin), macrollides (e.g., azithromycin, car pionic acid derivatives (e.g., alminoprofen, benoxaprofen, bomycin, clarithromycin, dirithromycin, erythromycin, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoX erythromycin acistrate, erythromycin estolate, erythromycin aprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, glucoheptonate, erythromycin lactobionate, erythromycin ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, propionate, erythromycin Stearate, josamycin, leucomycins, pirprofen, pranoprofen, protizinic acid, Suprofen, tiaprofenic 30 midecamycins, miokamycin, oleandomycin, primycin, roki acid, Ximoprofen, Zaltoprofen), pyrazoles (e.g., difenami tamycin, rosaramicin, roXithromycin, spiramycin, troleando Zole, epirizole), pyrazolones (e.g., apaZone, benZpiperylon, mycin), polypeptides (e.g., amphomycin, bacitracin, capreo feprazone, mofebutaZone, moraZone, oxyphenbutaZone, phe mycin, colistin, enduracidin, enviomycin, fusafungine, nylbutaZone, pipebuZone, propyphenaZone, ramifenaZone, gramicidin S. gramicidin(s), mikamycin, polymyxin, pristi SuxibuZone, thiazolinobutaZone), salicylic acid derivatives 35 namycin, ristocetin, teicoplanin, thiostrepton, tuberactino (e.g., acetaminosalol, aspirin, benorylate, bromosaligenin, mycin, tyrocidine, tyrothricin, Vancomycin, viomycin, Vir calcium acetylsalicylate, diflunisal, etersalate, fendosal, gen giniamycin, Zinc bacitracin), tetracyclines (e.g., apicycline, tisic acid, glycol salicylate, imidazole salicylate, lysine ace chlortetracycline, clomocycline, demeclocycline, doxycy tylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl cline, guamecycline, lymecycline, meclocycline, methacy salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, 40 cline, minocycline, Oxytetracycline, penimepicycline, pipa phenyl salicylate, Salacetamide, Salicylamide o-acetic acid, cycline, rollitetracycline, sancycline, tetracycline), and others salicylsulfuric acid, Salsalate, Sulfasalazine), thiazinecar (e.g., cycloserine, mupirocin, tuberin). boxamides (e.g., ampiroXicam, droxicam, isoxicam, lornoxi Other examples of antibiotics are the synthetic antibacte cam, piroxicam, tenoxicam), .epsilon.-acetamidocaproic rials, such as 2,4-diaminopyrimidines (e.g., brodimoprim, acid, S-(5'-adenosyl)-L-methionine, 3-amino-4-hydroxybu 45 tetroxoprim, trimethoprim), nitrofurans (e.g., furaltadone, tyric acid, amiXetrine, bendazac, benzydamine, alpha.-bis furazolium chloride, nifuradene, nifuratel, nifurfoline, abolol, bucolome, difenpiramide, ditazol, emorfaZone, fepra nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin), quino dinol, guaiaZulene, nabumetone, nimeSulide, oxaceprol, lones and analogs (e.g., cinoxacin, ciprofloxacin, clinafloxa paranyline, perisoxal, produaZone, Superoxide dismutase, cin, difloxacin, enoxacin, fleroxacin, flumequine, gatifloxa tenidap, Zileuton, their physiologically acceptable salts, com 50 cin, grepafloxacin, lomefloxacin, miloxacin, moxifloxacin, binations thereof, and mixtures thereof. nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic Non-limiting examples of antibiotics include doxorubicin; acid, paZufloxacin, pefloxacin, pipemidic acid, piromidic aminoglycosides (e.g., amikacin, apramycin, arbekacin, acid, roSoxacin, rufloxacin, sparfloxacin, temafloxacin, tosu bambermycins, butirosin, dibekacin, dihydrostreptomycin, floxacin, trovafloxacin), Sulfonamides (e.g., acetyl Sul fortimicin(s), gentamicin, isepamicin, kanamycin, microno 55 famethoxypyrazine, benzylsulfamide, chloramine-B, micin, neomycin, neomycin undecylenate, netilmicin, paro chloramine-T, dichloramine T. n-formylsulfisomidine, momycin, ribostamycin, Sisomicin, spectinomycin, Strepto n-beta-D-glucosylsulfanilamide, mafenide, 4'-(methylsul mycin, tobramycin, trospectomycin), amphenicols (e.g., famoyl)sulfanilanilide, noprylsulfamide, phthalylsulfaceta aZidamfenicol, chloramphenicol, florfenicol, thiampheni mide, phthalylsulfathiazole, Salazosulfadimidine, Succinyl col), ansamycins (e.g., rifamide, rifampin, rifamycin SV, rifa 60 sulfathiazole, Sulfabenzamide, Sulfacetamide, pentine, rifaximin), beta.-lactams (e.g., carbacephems (e.g., Sulfachlorpyridazine, Sulfachrysoidine, Sulfacytine, Sulfadi loracarbef)), carbapenems (e.g., biapenem, imipenem, mero azine, Sulfadicramide, Sulfadimethoxine, Sulfadoxine, Sulfa penem, panipenem), cephalosporins (e.g., cefaclor, ethidole, Sulfaguanidine, Sulfaguanol, Sulfalene, Sulfaloxic cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfame cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, 65 thizole, Sulfamethomidine, Sulfamethoxazole, Sulfamethoxy cefetamet, cefixime, cefinenoXime, cefodizime, cefonicid, pyridazine, Sulfametrole, Sulfamidochrysoidine, Sulfamox cefoperaZone, ceforamide, cefotaxime, cefotiam, cefoZop ole, Sulfanilamide, 4-Sulfanilamidosalicylic acid, US 9,289,396 B2 19 20 n-sulfanilylsulfanilamide, sulfanilylurea, n-sulfanilyl-3,4- prednisolone, methylprednisolone, prednisolone 21-phos Xylamide, Sulfanitran, Sulfaperine, Sulfaphenazole, Sul phate, prednisolone acetate, fluorometholone, betametha faproxyline, Sulfapyrazine, Sulfapyridine, Sulfasomizole, Sul Sone, fluocortolone, indomethacin and triamcinolone; decon fasymazine, Sulfathiazole, Sulfathiourea, Sulfatolamide, gestants such as phenylephrine, naphazoline and Sulfisomidine, Sulfisoxazole) Sulfones (e.g., acedapsone, ace tetrahydrazoline; miotics and anti-cholinesterase's Such as diasulfone, acetosulfone sodium, dapsone, diathymosulfone, pilocarpine, eserine salicylate, carbachol, di-isopropyl fluo glucosulfone sodium, Solasulfone, Succisulfone, Sulfanilic rophosphate, phospholine iodide, echothiophate, physostig acid, p-sulfanilylbenzylamine, Sulfoxone sodium, thiazolsul mine and demecarium bromide; mydriatics such as atropine fone), and others (e.g., clofoctol, hexedine, methenamine, Sulfate, cyclopentolate, homotropine, Scopolamine, tropica methenamine anhydromethylene citrate, methenamine hip 10 mide, eucatropine, and hydroxyamphetamine; sympathomi purate, methenamine mandelate, methenamine Sulfosalicy metics such as epinephrine and immunosuppressants such as late, nitroxoline, taurolidine, Xibomol). cyclosporin and azathioprine. Non-limiting examples of immunosuppressive agents Exemplary drugs for glaucoma treatment include beta include dexamethasone, cyclosporin A. azathioprine, brequi blockers (e.g., timolol, betaxolol, levobetaxolol, carteolol, nar, gusperimus, 6-mercaptopurine, mizoribine, rapamycin, 15 levobunolol, propranolol), carbonic anhydrase inhibitors tacrolimus (FK-506), folic acid analogs (e.g., denopterin, (e.g., brinzolamide and dorzolamide), alpha 1 antagonists edatrexate, methotrexate, piritrexim, pteropterin, TomudeX(R), (e.g., nipradolol), alpha2agonists (e.g. iopidine and brimoni trimetrexate), purine analogs (e.g., cladribine, fludarabine, dine), miotics (e.g., pilocarpine and epinephrine), prostaglan 6-mercaptopurine, thiamiprine, thiaguanine), pyrimidine din analogs (e.g., latanoprost (13.14-dihydro-17-phenyl-18, analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, 19,20-trinor-prostaglandin F2a-1-isopropyl ester), cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, travoprost, unoprostone, and compounds set forth in U.S. Pat. fluorouracil, gemcitabine, tegafur), fluocinolone, triami Nos. 5,889,052: 5,296,504; 5,422,368; and 5,151,444), nolone, anecortave acetate, fluorometholone, medrysone, and “hypotensive lipids” (e.g., bimatoprost and compounds set prednisolone. forth in U.S. Pat. No. 5,352.708), and neuroprotectants (e.g., Non-limiting examples of antifungal agents include poly 25 compounds from U.S. Pat. No. 4,690.931, particularly elip enes (e.g., amphotericin B, candicidin, dermostatin, filipin, rodil and R-eliprodil, as set forth in a pending application fungichromin, hachimycin, hamycin, lucensomycin, mepar U.S. Ser. No. 60/203,350 (PCT/US01/15074; PCT/US01/ tricin, natamycin, nystatin, pecilocin, perimycin), azaserine, 15169), and appropriate compounds from WO 94/13275, griseofulvin, oligomycins, neomycin undecylenate, pyirolini including memantine. trin, siccanin, tubercidin, viridin, allylamines (e.g., buten 30 Exemplary therapeutic agents with low solubility in aque afine, naftifine, terbinafine), imidazoles (e.g., bifonazole, ous solution for otic applications may also include epineph butoconazole, chlordantoin, chlormidazole, cloconazole, rine, steroids, certain antibiotics as described above, and neu clotrimazole, econazole, enilconazole, fenticonazole, flutri rotrophins. Other exemplary drugs include, but are not mazole, isoconazole, ketoconazole, lanoconazole, micona limited to, Auralagan (benzocaine plus antipyrine), Ciprodex Zole, omoconazole, oxiconazole nitrate, Sertaconazole, Sul 35 (ciprofloxacin and dexamethasone), Cortisporin (hydrocorti conazole, tioconazole), thiocarbamates (e.g., tolciclate, Sone, polymiyxin, and neosporin), Triethanolamine, and tolindate, tolnaftate), triazoles (e.g., fluconazole, itracona Ofloxacin (Floxin). Zole, Saperconazole, terconazole), acrisorcin, amorolfine, Exemplary therapeutic agents for sinus applications biphenamine, bromosalicylchloranilide, buclosamide, cal include, but are not limited to, (1) steroids selected from the cium propionate, chlorphenesin, ciclopiroX, cloxyquin, 40 group consisting of beclomethasone, flunisolide, fluticaSone, coparaffinate, diamthazole dihydrochloride, exalamide, triamcinolone, mometasone, aclometaSone, desonide, hydro flucytosine, halethazole, hexetidine, loflucarban, nifuratel, cortisone, betamethasone, clocortolone, desoximetaSone, potassium iodide, propionic acid, pyrithione, salicylanilide, fluocinolone, flurandrenolide, mometaSone, prednicarbate; Sodium propionate, Sulbentine, tenonitrozole, triacetin, amcinonide, desoximetasone, diflorasone, fluocinolone, ujothion, undecylenic acid, and Zinc propionate. 45 fluocinonide, halcinonide, clobetasol, augmented betametha Non-limiting examples of antiviral agents include acyclo Sone, diflorasone, halobetasol, prednisone, dexamethasone Vir, carbovir, famciclovir, ganciclovir, penciclovir, and and methylprednisolone; (2) antimicrobials selected from the Zidovudine. group consisting of amantadine, amrimantadine, oselta Non-limiting examples of antiprotozoal agents include mivir, Zanamivir, inoglycosides, amikacin, tobramycin, pentamidine isethionate, quinine, chloroquine, and meflo 50 amoxicillin, amoxicillin/clavulanate, amphotericin B, ampi quine. cillin, amplicillin/Sulbactam, atovaquone, azithromycin, cefa More broadly speaking, ophthalmic drugs include without Zolin, cefepime, cefotetan, cefpdoXime, ceftazidime, cefiri limitation antibiotics, anti-fungals, anti-virals, anti-inflam axone, cefuroxime, cefhroXime axetil, chioramphenicol, matories, anti-glaucoma, anti-VEGF, and the like. Some spe clotrimazole, ciprofloxacin, clarithromycin, dapsone, doxy cific examples of ophthalmic drugs include antibiotics Such 55 cycline, erythromycin, fluconazole, foScarnet, ganciclovir, as tetracycline, chlortetracyline, bacitracin, neomycin, poly atifloxacin, imipenem/cilastatin, itraconazoic, ketoconazole, myxin, gramicidin, oxytetracycline, chloramphenicol, genta metronidazole, nystatin, penicillins including penicillin G, mycin, penicillin, kanamycin, amikacin, Sisomicin, tobramy pentamidine, piperacillin/taZobactam, rifampin, quinupris cin, garamycin, ciprofloxacin, norfloxacin and erythromycin; tin-dalfopristin, ticarcillin/clavulanate, trimethoprim/sul antibacterials such as Sulfonamides, Sulfacetamide, Sulfame 60 famethoxazole, Valacyclovir, mafenide, silver Sulfadiazine, thizole and Sulfisoxazole; antivirals, including idoxuridine; mupirocin, nystatin, triameinolone/nyStatin, clotrimazole/be and other antibacterial agents such as nitrofuraZone and tamethasone, clotrimazole, ketoconazole, butoconazole, Sodium propionate; anti-allergenics such as antazoline, meth miconazole, tioconazole, detergent-like chemicals that dis apyriline, chlorpheniramine, pyrilamin and prophenpy rupt or disable microbes, menfegol, and N-docasanol, chemi ridamine; anti-inflammatories such as cortisone, hydrocorti 65 cals that block microbial attachment to target cells, chemicals SOne, hydrocortisone acetate, dexamethasone, that inhibits entry of infectious pathogens into cells, Sul dexamethasone 21-phosphate, fluocinolone, medrysone, phated polymers, Sulponated polymers, antiretroviral agents, US 9,289,396 B2 21 22 agents which change the condition of the tissue to make it compound achieves a certain concentration at a specific site, hostile to the microbes, agents which alter mucosal pH, buffer reducing possible inaccuracy due to mistaken dose or gel, acidform non-pathogenic microbes that kill or inhibit the improper placement. growth of pathogenic microbes and antimicrobial metals. Specific Ocular Pathology: Glaucoma Other exemplary agents include corticosteroid or other 5 Glaucoma is a leading cause of blindness worldwide. anti-inflammatory drugs as noted above (e.g., an NSAIDs), While the exact etiology is unknown, there is either overpro decongestants (e.g., vasoconstrictor), mucous thinning duction or impaired exit of aqueous humor from the eye agents (e.g., an expectorant or mucolytic), agents that pre causing elevated intraocular pressure (IOP). Elevated IOP vents or modifies an allergic response (e.g., an antihistamine, causes damage to the optic nerve and, ifuntreated, can result cytokine inhibitor, leucotriene inhibitor, IgE inhibitor, immu 10 in loss of vision. nomodulator), anesthetic agents with or without a vasocon There are many sub-types of glaucoma. The initial treat striction agents (e.g. Xylocaine with or without Epinephrine), ment is usually topical medication to lower the IOP by either analgesic agents, hemostatic agents to stop bleeding, anti reducing aqueous production or increasing aqueous outflow proliferative agents, cytotoxic agents e.g. stem cells, genes or through the trabecular meshwork or uveal-scleral channels. gene therapy preparations, viral vectors carrying proteins or 15 Oral medication is also available but rarely used on a long nucleic acids such as DNA or mRNA coding for important term basis due to side effects. Surgery is performed when therapeutic functions or Substances, cauterizing agents e.g. medical treatments do not control the IOP. silver nitrate, etc. Ocular hypertension is a condition wherein intraocular In general, drugs are added to drug reservoir films or film pressure is elevated but no apparent loss of visual function has layers at concentrations Suitable to provide Sustained release, occurred; Such patients are considered to be at significant risk and in some embodiments, controlled release, for appropriate for the eventual development of the visual loss associated therapeutic activity for desired delivery time periods. For with glaucoma. If ocular hypertension is detected early and example, as described in the Examples, Latanoprost (Xala treated promptly with medications that effectively reduce tan(R), a medication that reduces the pressure inside the eye, elevated intraocular pressure, loss of visual function or its can be added as an ethanol solution to reconstituted, deriva 25 progressive deterioration can generally be ameliorated. tized collagen solutions to produce a film construct providing Collagen-based drug delivery provides a simple and effec at least a 3-month daily delivery of at least about 1.5 lug/day. tive means of delivering pressure-lowering agents directly to Other examples are provided in the Detailed Disclosure and the affected site to treat glaucoma and ocular hypertension. Examples. Examples of agents used for treating glaucoma and ocular Other drug reservoir films or film layers can similarly be 30 hypertension that can be delivered using the collagen-based prepared to provide delivery of therapeutically effective of constructs described herein include: beta-blockers (e.g., amounts of each drug. In some embodiments, the delivery timolol, betaxolol, levobetaxolol, carteolol, levobunolol, pro systems are prepared by forming films or membranes of acy pranolol); carbonic anhydrase inhibitors (e.g., dorzolamide); lated collagen containing extended therapeutic concentra alpha 1 antagonists (e.g.); alpha 2 agonists (e.g. iopidine tions of selected drugs and exposing the drug containing 35 and brimonidine), miotics (e.g., pilocarpine and phosphodi compositions to ultraviolet irradiation in a nitrogen or other esterade inhibitors; sympathomimetics (e.g., epinephrine), oxygen depleted atmosphere for time periods ranging from prostaglandin analogs (e.g., travoprost, unoprostone, and about 14 to about 19 minutes, about 15 to about 19 minute, compounds set forth in U.S. Pat. Nos. 5,889,052; 5.296,504; about 16 to about 18 minutes, about 15, about 16, about 17, 5,422.368; and 5,151,444), “hypotensive lipids” (e.g., about 18, or about 19 minutes. In some embodiments, the 40 bimatoprost and compounds set forth in U.S. Pat. No. 5,352. exposure time is approximately 18 minutes. 708), and neuroprotectants (e.g., compounds from U.S. Pat. Exposure to ultraViolet irradiation in a nitrogen (or other No. 4,690,931, particularly eliprodil and R-eliprodil, as set oxygen free) atmosphere is conducted to produce collagen forth in U.S. Ser. No. 60/203,350 (PCT/US01/15074; PCT/ films, wafers, or membranes with defined resorption charac US01/15169), and appropriate compounds from WO teristics ranging from greater than about 1 month, 2 months, 45 94/13275, including memantine. 3 months, 4 months, 5 months, to about 6 months, or to greater Under local or topical anesthesia, the collagen-based drug than 6 months, the resorption characteristics being controlled delivery construct containing a pressure lowering medication by time of exposure to ultraviolet light in the oxygen free would be surgically implanted under the conjunctiva or ten atmosphere. For example, exposure for about 18 minutes (or on's capsule approximately 3 mm posterior to the limbus in from 17 to 19 minutes) can be used to produce films or 50 between the rectus muscles. It would provide long term, Zero membranes resorbing or degrading after about 6 months. In order or near Zero order drug delivery in a therapeutic amount Some embodiments, the release is for greater than 6 months but require essentially no patient involvement. Medication but for less than one year; for greater than 6 months but for would be released and diffuse through the conjunctiva into the less than 9 months; for greater than 6 months but for less than tear film constantly bathing the cornea and penetrating into 8 months; or for greater than 6 months but for less than 7 55 the eye. In addition, an episcleral placement of the collagen months. Other exemplary release profiles for sustained and/or based drug delivery construct would be directly over the controlled release are described herein. ciliary body, trabecular meshwork and Schlems canal, which The present invention provides a number of advantages. are the target sites for anti-glaucomatherapy. Thus, episcleral For example, the present techniques and collagen film com placement will also allow trans-scleral diffusion to these tis positions facilitate an improved approach for Sustained, and 60 Sues, an advantage not found with topical medication. in some embodiments, controlled delivery of active agents Accordingly, the drug delivery systems and constructs where a precise dose and accurate placement are required. described herein provide methods of treating glaucoma and The dose can be adjusted to any desired amount, i.e., by ocular hypertension. modifying the concentration of compound in the film or the Post Operative Drug Delivery size of the film, and the solid nature of the film allows its 65 Another situation in which the collagen-based drug deliv placement at any site in the body which can be reached by ery constructs can be used is following Surgery. Surgeons Surgical techniques. These features ensure that a delivered routinely prescribe medications after all types of Surgery. For US 9,289,396 B2 23 24 example, in almost every case, an ophthalmic Surgeon will ics. Most people recover fully when treated with an antibiotic prescribe topical antibiotics as prophylaxis against infection. for acute sinusitis that is caused by a bacterial infection. Topical corticosteroids and/or topical non-steroidal anti-in Sinusitis that lasts 8 weeks or longer is called chronic flammatory drugs (NSAID) are also routinely dispensed and sinusitis. It is more difficult to treat and responds more slowly are typically used at least four times per day. It is often to antibiotics than acute sinusitis. Antibiotic therapy is usu difficult for patients to administer these medications during ally recommended for chronic sinusitis and may require a the post operative period due to tissue Swelling, increased longer course of treatment and multiple antibiotics. A corti tearing and irritation. A passive drug delivery system, placed costeroid that reduces inflammation and Swelling of the lining under the conjunctiva or tenon's capsule by the Surgeon at the of the nasal passages may also be used during treatment. conclusion of the case, would eliminate the need for patients 10 In some people, a sinus infection may be caused by a to administer drugs post-operatively. Thus, the drug delivery fungus or a bacterium other than those normally associated systems and constructs described herein provide methods of with sinusitis. People who have an impaired immune system treating or preventing post-operative infection and other post are at risk for these unusual infections. It also may include operative complications. 15 people who must use oral or inhaled corticosteroid medica The subconjunctival fluids will cause the collagen in the tions. Fungal sinusitis, which accounts for a significant num device to hydrolyze thereby continuously releasing drugs in ber of chronic sinusitis cases, does not respond to antibiotic the Sub-Tenon's or Subconjunctival space. As the drugs are treatment and may need treatment with antifungal medica freed, they would diffuse through the conjunctiva and into the tions, corticosteroids, or Surgery. tear film constantly bathing the cornea and penetrating into Surgery may be required upon failure of antibiotics for an the eye. Furthermore, since the device is placed on the epis extended period of time. Symptoms of chronic sinusitis are clera, some of the drugs will diffuse directly into the eye often vague and may not respond well to treatment. It may through the Sclera; this is an advantage not found with topical take time and patience to find a Successful treatment. Thus, a application. After a predetermined time, the collagen delivery durable treatment modality would serve this patient popula device will completely disappear. 25 tion exceedingly well. Sinusitis Treatment Oral antibiotics are one of the most prescribed treatments Collagen-based drug delivery constructs can also be used for chronic sinusitis patients. For this treatment, doctors pre to deliver drugs directly into the sinuses, which area frequent scribe a number of broad-spectrum antibiotics that a patient site of infection for the treatment of sinusitis. They are closed, takes daily for approximately 2-3 weeks. difficult to access, and provide a very hospitable environment 30 Nebulized antibiotics are a means of treating infections for organisms to thrive. More than 37 million Americans topically. This treatment consists of breathing in the antibi suffer from at least one episode of acute sinusitis each year. otics through the nose, which allows the antibiotics to get This represents approximately 1 of 7 Americans. The preva directly to the source of the problem. In select cases, intrave lence of sinusitis has Soared in the last decade possibly due to nous antibiotic therapy is the method of choice. increased pollution, urban sprawl, and increased resistance to 35 Some currently utilized agents are: Antibiotics, such as antibiotics. For acute sinusitis, symptoms include facial pain/ Amoxil (Amoxacillin), Augmentin (amoxacillin-clavulate), pressure, nasal obstruction, nasal discharge, diminished Avelox (Moxifloxacin HCl), Bactrim (Sulfamethoxazole sense of Smell, and cough not due to asthma (in children). Trimethoprim), Biaxin (clarithryomycin), Cipro (ciprofloxa Additionally, sufferers of this disorder can incur fever, cin), AVelox (moxifloxacin), Doxycycline, Levaquin (levof fatigue, dental pain, and cough. 40 loxacin), Tequin (gatifloxacin), Zithromax (azithromycin); Sinusitis is an infection of the sinuses or inflammation of Decongestents, such as, Claritin (loratadine), ClarineX the sinuses. Infection is frequently a result of bacterial, viral (desloratadine), Allegra (fexofenadine), Zyrtec (cetirizine or fungal etiologies. Streptococcus pneumonia is the most HCL), Chlor-Trimeton (chiorpheneramine maleate); and common cause of acute infections of the sinuses. When there other drugs, such as corticosteroids, antihistamines, or pseu is an infection, there is typically sinus pressure on both cheeks 45 doephedrine. and heaviness in sinuses accompanied with chronic cough on Sinus Surgery is generally a last line of defense for doctors occasion. In many patients, the chronic cough is a result of to relieve a chronic sinusitis condition. Most Surgeries are acute sinusitis. Sinusitis also presents with post nasal drip endoscopic Surgeries, which allows the Surgeon to operate with a low grade fever and chills. without making facial incisions. The Surgery is generally Other bacteria involved in acute sinusitis include H. influ 50 accomplished in one to three hours, and can take several enza bacteria, which is more common in children under six weeks for a full recovery. years of age, and catarrhalis bacteria infection. Patients with A durable mechanism of drug delivery to the sinuses would nasal allergies can develop sinus infections due to a blockage be a welcome addition to the armamentarium of the treating of the opening of the sinuses into the nasal cavity. The eth physicians. A pharmaceutical agent, when afforded a longer moid sinus, maxillary sinus and frontal sinus all drain into the 55 resident time in this hostile environment, would have a nasal cavity. These become blocked as a result of the swelling greater opportunity to Successfully eradicate the organisms as of mucus membrane. well as prevent any disease recurrence. Thus, collagen-based Sinus infections have very high recurrence rates. Another drug delivery constructs in conjunction with the appropriate contributing factor to the severity of sinus infections is the antibiotics, antiviral agents, anti-fungal agents, anti-inflam fact that the sinuses afford an additional layer of protection to 60 matory agents, and other remedies will provide a very effec the harbored organisms, and they are thus very difficult to tive means for preferential treatment of bacterial, fungal, and eradicate. Again these facts would indicate that a better means viral sinus infections. of targeting delivery would be more effective. The constructs can be directly inserted into one or more of Short-term (acute) sinusitis usually lasts less than 4 weeks. a naturally occurring or man-made anatomical cavity or pas Up to two-thirds of people with acute sinusitis improve with 65 sageway Such as a nostril nasal cavity, meatus, interior of out antibiotic treatment. Sinus infections are commonly sinus, etc. to deliver atherapeutic Substance to tissues located caused by viral infections, which do not respond to antibiot adjacent to or near the implanted device US 9,289,396 B2 25 26 As with constructs prepared for optic use, constructs for therapy increases compliance but should be used only when sinus use are prepared by adding drugs to drug reservoir films equal clinical efficacy can be assured. In many instances, or film layers at concentrations suitable to provide Sustained palatability ultimately determines compliance in children. release, and in Some embodiments, controlled release, for Most cases of otitis media externa occur after an episode of appropriate therapeutic activity for desired delivery time peri acute otitis media, and 67% of patients develop a middle ear ods. In some embodiments, the delivery systems are prepared effusion. The mean duration of the effusions is 23 days, but by forming films or membranes of acylated collagen contain many persist much longer. Most cases of chronic otitis media ing extended therapeutic concentrations of selected drugs and are associated with conductive hearing loss, averaging exposing the drug containing compositions to ultraviolet irra approximately 25 dB. The following are among the many diation in a nitrogen (or other oxygen free) atmosphere for 10 strategies advocated for medical treatment in patients with time periods ranging from about 14 to about 19 minutes, Otitis media externa: antimicrobials; antihistamine-decon about 15 to about 19 minutes, about 16 to about 18 minutes, gestants, intranasal and systemic steroids, nonsteroidal anti about 15, about 16, about 17, about 18, or about 19 minutes. inflammatory drugs (NSAIDs), mucolytics, and aggressive In Some embodiments, the exposure time is approximately 18 management of allergic symptoms. minutes. Exposure to ultraviolet irradiation in a nitrogen (or 15 other low oxygen) atmosphere is conducted to produce col Controversy continues over the optimal management of lagen films or membranes with defined resorption character Otitis media. The current therapeutic regimens of tubes and istics as described herein, for example, ranging from greater antibiotics seem very inadequate in many cases. Clearly a than about 1 month, 2 months, 3 months, 4 months, 5 months, more durable treatment would be optimal so that here, too, a to about 6 months, the resorption characteristics being con collagen-based drug delivery construct would afford patients trolled by time of exposure to ultraviolet light in an oxygen a much more effective and durable treatment. The construct free atmosphere. can be inserted through a preexisting opening that was ulti Otitis Media Therapy lized for the placement of tubes. Inner ear tissue structures Another application for collagen-based drug delivery con that may be targeted for treatment purposes include but are structs is in drug delivery into the middle ear for the treatment 25 not limited to the cochlea, the endolymphatic Sac/duct, the of otitis media. The middle ear is has some of the same vestibular labyrinth, and all of the compartments (and con resistant characteristics as the sinuses. Otitis media is an necting tubes) which include these components. Access to inflammation and/or infection of the middle ear. Acute otitis these and other inner ear tissue regions is typically achieved media (acute ear infection) occurs when there is bacterial or through a variety of structures, including but not limited to the viral infection of the fluid of the middle ear, which causes 30 round window membrane, the oval window? stapes footplate, production of fluid or infection. Chronic otitis media occurs the annular ligament, and the otic capsule/temporal bone, all when the eustachian tube becomes blocked repeatedly due to of which are considered "middle-inner ear interface tissue allergies, multiple infections, ear trauma, or Swelling of the structures.” Furthermore, as used herein, "middle ear” means adenoids. the physiological air-containing tissue Zone behind the tym Otitis media is the second most common disease of child 35 panic membrane (e.g. the ear drum) and ahead of the inner ear. hood, after upper respiratory infection. It is also the most Once again, the construct will have a protracted resident common cause for childhood visits to a physicians office, time in the middle ear, and thereby will be more durable. This accounting for approximately 20 million annual physician durability will provide a more effective treatment against the visits. Various epidemiologic studies report the prevalence pathological organisms, as well as decrease the possibility of rate of acute Otitis media to be 17-20% within the first 2 years 40 recurrent disease because of its ability to more completely of life, and 90% of children have at least one documented eradicate the pathological organisms. middle ear effusion by age 2 years. Acute Otitis media is a As with constructs prepared for optic or sinus use, con recurrent disease. More than one third of children experience structs for otic use are prepared by adding drugs to drug 6 or more episodes of acute Otitis media by age 7 years. reservoir films or film layers at concentrations suitable to Chronic Suppurative Otitis media is a chronic inflamma 45 provide Sustained release, and in some embodiments, con tion of the middle ear that persists at least 6 weeks and is trolled release, for appropriate therapeutic activity for desired associated with otorrhea through a perforated TM, an ind delivery time periods. In some embodiments, the delivery welling tympanostomy tube or a Surgical myringotomy. Mor systems are prepared by forming films or membranes of acy bidity from this disease remains significant, despite frequent lated collagen containing extended therapeutic concentra use of systemic antibiotics to treat the illness and its compli 50 tions of selected drugs and exposing the drug containing cations. Intratemporal and intracranial complications of otitis compositions to ultraviolet irradiation in a nitrogen atmo media are the 2 major types. Intratemporal complications sphere for time periods ranging from about 14 to about 19 include hearing loss (conductive and sensorineural), tym minutes, about 15 to about 19 minutes, about 16 to about 18 panic Membrane perforation (acute and chronic), chronic minutes, about 15, about 16, about 17, about 18, or about 19 Suppurative otitis media (with or without cholesteatoma), 55 minutes. In some embodiments, the exposure time is approxi cholesteatoma, tympanosclerosis, mastoiditis, petrositis, mately 18 minutes. Exposure to ultraviolet irradiation in a labyrinthitis, facial paralysis, cholesterol granuloma, and nitrogen (or other oxygen free) atmosphere is conducted to infectious eczematoid dermatitis. Intracranial complications produce collagen films or membranes with defined resorption include meningitis, Subdural empyema, brain abscess, extra characteristics as described herein, for example, ranging from dural abscess, lateral sinus thrombosis, and otitic hydroceph 60 greater than about 1 month, 2 months, 3 months, 4 months, 5 alus. months, to about 6 months, or to greater than 6 months, the Compliance, duration of therapy, and cost are important resorption characteristics being controlled, at least in part, by issues in treating children with acute otitis media. The pri time of exposure to ultraviolet light in a nitrogen atmosphere. mary determinants of compliance appear to be frequency of The features and other details of the invention will now be dosing, palatability of the agent, and duration of therapy. Less 65 more particularly described and pointed out in the following frequent doses are more desirable than more frequent doses, examples describing preferred techniques and experimental which interfere with daily routines. Shorter duration of results. These examples are provided for the purpose of illus US 9,289,396 B2 27 28 trating the invention and should not be construed as limiting, ing from 1 day to 27 days and replaced with 1.0 mL of fresh particularly with respect to the exemplified drug. buffer. Latanoprost concentration in buffer aliquots was mea sured by HPLC analysis (Millennium Research Laborato EXAMPLES ries). Detection at nanogram levels was validated using Latanoprost standards and concentration curves generated. Example 1 In the second experiment, four 8 mm diameter polymerized films/wafers were placed in 1.5 mL vials containing 1 mL of Sustained Release from Multi-directional 0.1 M phosphate buffer, pH 7.4. The vials were placed in a Collagen-based Films controlled temperature chamber at 37°C. Two of the films/ 10 wafers were exposed to UV irradiation for 14 minutes. The Preparation of Collagen second set offilms/wafers were exposed to UV irradiation for Purified, pepsin digested collagen at 3 mg/mL was 18 minutes. At various time intervals from day 1 to day 180 obtained from Advanced Biomatrix, Inc. and derivatized with the entire 1.0 mL of buffer was removed from the vials and glutaric anhydride as previously described (U.S. Pat. Nos. replaced with fresh buffer. Collected buffer solution was then 5,631,243 and 5.492,135, incorporated by reference). Briefly, 15 assayed for Latanoprost using an EIA kit (Cayman Chemi the collagen solution was adjusted to pH 9.0 with 10 N and 1 cal). Buffer has continued to be removed from the 18 minute N NaOH. While stirring the solution, glutaric anhydride was UV exposed Latanoprost samples out to more than 240 days. added at 10% (weight of collagen). For 5 minutes, the stirring The second set of films/wafers exposed to UV irradiation for continued, and the pH was maintained. 14 minutes began to degrade after 180 days. Initial results are The pH of the solution was then adjusted to 4.3 with 6 N presented below. Additional assay results for Latanoprost and 1 N HCl to precipitate the derivatized collagen. The concentration is pending. precipitate was centrifuged at 3500 rpm for 20 minutes and In a third experiment, three pretreated 8 mm diameter washed one time in pyrogen-free deionized water and then polymerized films/wafers containing 250 ug of Latanoprost redissolved in phosphate buffer (0.01 Mphosphate buffer, pH per wafer were placed in 1.5 mL vials containing 1 mL of 0.1 7.4) to achieve a final concentration of approximately 30 25 M phosphate buffer, pH 7.4. The vials were placed in a con mg/ml. trolled temperature chamber at 37°C. At various time inter Preparation of Collagen Delivery Systems Containing vals from day 1 (after pretreatment) to day 70, the entire 1.0 Latanoprost mL of buffer was removed from the vials and replaced with To prepare Latanoprost (13,14-dihydro-17-phenyl-18, 19. fresh buffer. Collected buffer solution was then assayed for 20-trinor-prostaglandin F2a-1-isopropyl ester) collagen films 30 Latanoprost using an EIAkit (Cayman Chemical). Buffer has or wafers, 50 L of Latanoprost in ethanol at 50 ug/uL was continued to be removed from the Latanoprost samples out to mixed with 1 mL of derivatized collagen described above. more than 140 days. Initial assay results are presented below. The mixture was centrifuged at 3500 rpm for 10 minutes if air Additional assay results of Latanoprost release from day 70 to bubbles were observed. 100 uL aliquots of the mixture con day 148 for the pretreated films/wafers is pending. taining 250 ug of Latanoprost were then Syringed onto 8 mm 35 Both in situ polymerizing collagen gels and solid collagen diameter polypropylene molds. The molds were placed in a films/wafers produced Sustained release of Latanoprost. laminar flow hood until the concentrated collagen plus HPLC analysis of release from the polymerizing gels showed Latanoprost was dry. a large initial release of Latanoprost from polymerizing gels A. Polymerized Collagen Films/Wafers added directly to buffer solution followed by sustained The dried collagen films or wafers were placed in a con 40 release averaging 1.4 ug/day. (FIG. 1, set 1.) Polymerizing trolled atmosphere chamber containing an ultraviolet lamp gels partially pre-polymerized before placing in buffer solu emitting 254 nm of irradiation. Prior to activating the ultra tion showed reduced initial release followed by sustained violet lamp, the chamber was flushed with nitrogen gas. After release averaging 1.68 ug/day. (FIG. 1, set 2.) Release from 5 minutes of flushing, the exit port was closed, the ultraviolet gels was near steady state following a large bolus release of up lamp activated and the collagen composition exposed to 254 45 to day 3. (FIG. 1). nm ultraviolet irradiation for 14-18 minutes while continuing Release from UV polymerized films/wafers was measured to flush the chamber to maintain chamber expansion. The using the Latanoprost EIA kit from Cayman Chemical Com resulting materials are referred to herein as “polymerized pany following kit instructions out to 180 days. (FIG. 2.) The films/wafers.” pretreated UV polymerized films/wafers (pre-treated in B. "Pretreated Collagen Films/Wafers” 50 buffer for 7-10 days) had a nearly steady state release for the Another set of film/wafers were prepared as described 70 days measured following pretreatment. (FIG. 3.) above. In this case, however, the polymerized films/wafers Degradation of Collagen Films/Wafers containing 250 ug of Latanoprost per wafer were pretreated in During in vitro incubation in buffer solution, the in situ 0.1M phosphate buffer for 7 days to permit initial bolus polymerizing collagen gels remained present throughout the release of drug prior to use in experiments. The preparations 55 27-day testing period and beyond. In vitro incubation of col are referred to as the “pretreated films/wafers’. lagen films/wafers containing Latanoprost remained intact C. In Situ Polymerizing Collagen Gels throughout the testing period noted above. Films/wafers In situ polymerizing collagen gels was prepared by exten exposed to UV irradiation for 14 minutes began to degrade sive dialysis of salt precipitated collagen against 0.035 M after 180 days. Films/wafers exposed to UV irradiation for 18 EDTA with step-wise increase of pH to 7.5. Latanoprost was 60 minutes appeared intact even after 248 days. Pretreated films/ dissolved in ethanol and added to polymerizing collagen gels wafers appeared intact after 145 days of incubation. to provide gel depots containing 250 ug of Latanoprost/100 Therapeutic Potential of Latanoprost Released from Collagen uL collagen gel. Films Evaluation of Latanoprost Release In Vitro Results of Latanoprost release experiments demonstrate In the first experiment, collagen gels containing Latano 65 the potential of providing a durable implant for Sustained prost were incubated in 1.0 mL of physiologic saline at 37°C. release of Latanoprost for effective treatment of glaucoma. The entire 1.0 mL volume was removed at time periods rang Although both in situ polymerizing collagen gels and the US 9,289,396 B2 29 30 collagen wafers/films could be used in the treatment of glau buffer (0.01 M phosphate buffer, pH 7.4) to achieve a final coma if such collagen depots were injected (gel) or implanted concentration of approximately 30 mg/ml. (wafer) in, for example, the Subconjunctival space, the pre Preparation of Uni-directional Collagen Films Containing treated wafers provided a sustained release without the initial Latanoprost burst observed with the polymerizing gel. Based on the sus To prepare Latanoprost uni-directional collagen films, 50 tained release observed through day 180, it is anticipated that ug of Latanoprost ethanol Solution containing 50 ug/uL is the collagen wafers can be used to deliver Latanoprost for a mixed with 1 mL of glutaric anhydride-derivatized collagen period of at least 6 months following each implantation, and (reservoir layer) described above. The mixture is centrifuged even longer delivery periods may be feasible. at 3500 rpm for 10 minutes if air bubbles were observed. Pharmacokinetic Study in Rabbits 10 Aliquots of 100LL are syringes onto 8 mm diameter polypro A Stage 1 PK study was conducted in the rabbit model. Six pylene molds or onto plates. The molds/plates are placed in a animals were included in this preliminary PK study. Collagen laminar flow hood until the collagen plus Latanoprost is par films/wafers were fabricated as described above and cut into tially dried, appearing slightly gelatinous. A layer of barrier circular shapes of approximately 5 mm or oval shapes 15 film collagen composition is carefully placed on the Surface approximately 4 mm wide by 3 mm long. Latanoprostwafers of the reservoir layer and the wells placed again in the sterile were loaded with either 100 ug of Latanoprost/wafer or 200 laminar flow hood to fully dehydrate the unidirectional film. ug of Latanoprost/wafer. The conjunctival tissue was pre The dried collagen film is placed in a controlled atmo pared for wafer insertion by exposing the Superior-temporal sphere chamber containing an ultraviolet lamp emitting 254 bulbar conjunctival area in an area approximately 1 mm away nm of irradiation. Prior to activating the ultraviolet lamp, the from the corneal limbus and dissecting the conjunctiva with chamber is flushed with nitrogen gas or otherwise rendered Scissors under a Surgical microscope. Wafers were placed into oxygen free. After 5 minutes of flushing, the exit port is the conjunctival incision. In some cases the wafers were closed, the ultraviolet lamp activated and the collagen com trimmed before insertion. Seven days following wafer inser position exposed to 254 nm ultraviolet irradiation for 18 tion, wafers were removed and the following tissues and 25 minutes while continuing to flush the chamber to maintain fluids were removed for testing: aqueous fluid, vitreous, iris/ chamber expansion. ciliary tissue, and inferior and posterior conjunctival tissue. Planned Evaluation of Latanoprost Diffusion Though Barrier Aqueous samples were assayed for Latanoprost acid using Layer the EIA kit from Cayman Chemical Company. Latanoprost Polymerized films are cut to tightly fit on the filter of 2 mL, acid was detected in all rabbit aqueous samples. However, 30 nonsterile, 0.45 um Nylon Membrane, Centrifugal Filter results were complicated by possible interference from Tubes. Buffer is placed on the reservoir side of the film and endogenous IgG and IgE antibodies in the rabbit aqueous. placed in a 37°C. incubator for 1 hour, 6 hours, 24 hours, and Final results are pending. 48 hours. Aliquots of the buffer passing through the film/filter are analyzed for Latanoprost. Control films without barrier Example 2 35 film are also tested. Latanoprost concentration is measured as described in Experiment 1. It is anticipated that the diffusion Sustained Release from Uni-directional experiments will demonstrate the effectiveness of the barrier Collagen-based Films layer in preventing Latanoprost diffusion through the barrier layer, thus providing uni-directional films to enhance the Reservoir Layer: 40 effectiveness of drug release. Purified, pepsin digested collagen at 3 mg/mL is obtained Although the present invention has been described with from Advanced Biomatrix, Inc. and derivatized with glutaric reference to exemplary embodiments, one skilled in the art anhydride as previously described (U.S. Pat. Nos. 5,631.243 can easily ascertain its essential characteristics and without and 5.492.135). Briefly, the collagen solution is adjusted to departing from the spirit and scope thereof, can make various pH 9.0 with 10 N and 1 N. NaOH. While stirring the solution, 45 changes and modifications of the invention to adapt it to glutaric anhydride is added at 10% (weight of collagen). For various usages and conditions. Those skilled in the art will 5 minutes, the stirring is continued, and the pH maintained. recognize or be able to ascertain using no more than routine The pH of the solution is then adjusted to 4.3 with 6 N and 1 experimentation, many equivalents to the specific embodi NHCl to precipitate the derivatized collagen. The precipitate ments of the invention herein. Such equivalents are intended is centrifuged at 3500 rpm for 20 minutes and washed one 50 to be encompassed in the scope of the present invention. time in pyrogen-free deionized water and then redissolved in All references, including patents, publications, and patent phosphate buffer (0.01 M phosphate buffer, pH 7.4) to applications, mentioned in this specification are herein incor achieve a final concentration of approximately 30 mg/ml. porated by reference in the same extent as if each independent Barrier Layer: publication, patent or patent application was specifically and Purified, pepsin digested collagen at 3 mg/mL is obtained 55 individually indicated to be incorporated by reference. from Advanced Biomatrix, Inc. and derivatized with glutaric anhydride and B-styrene sulfonyl chloride as previously We claim: described (U.S. Pat. No. 5,480,427). Briefly, the collagen 1. A collagen-based, ultraviolet radiation crosslinked, film, solution is adjusted to pH 9.0 with 10 N and 1 NNaOH. While wafer, or membrane construct comprising at least one layer of stirring the solution, glutaric anhydride is added at 10% 60 chemically derivatized collagen and an amount of an oph (weight of collagen). For 5 minutes, the stirring is continued, thalmic, otic, or nasal drug that has low water or aqueous and the pH maintained and 2% B-styrene sulfonyl chloride solubility, wherein the amount of the drug is sufficient to added at pH 9.0 and stirred for another 10 minutes. The pH of provide a therapeutically effective amount of drug per day the solution is then adjusted to 4.3 with 6 N and 1 NHCl to over a period of at least three months, and further wherein the precipitate the derivatized collagen. The precipitate is centri 65 construct has been exposed to ultraviolet radiation crosslink fuged at 3500 rpm for 20 minutes, washed one time in pyro ing for a period of 17 to 19 minutes in a low oxygen environ gen-free deionized water and then redissolved in phosphate ment. US 9,289,396 B2 31 32 2. The collagen-based film, wafer, or membrane of claim 1, (ii) dissolving the derivatized collagen precipitates in wherein the drug present in the film, wafer, or membrane is physiological buffer at concentrations ranging from 10 resistant to removal by a 1 day treatment in an aqueous buffer. mg/mL to 100 mg/mL: 3. The collagen-based film, wafer, or membrane of claim 2, (iii) adding active drug in an amount and concentration wherein the drug present in the film, wafer, or membrane is Sufficient to provide sustained release at therapeutic lev resistant to removal by a 7 day treatment in an aqueous buffer. els for at least three months; 4. The film, wafer, or membrane of any one of claims 1 to (iv) casting the collagen-drug solution into a thin layer or 3, further comprising a barrier layer of collagen derivatized onto a mold; by an acylation agent. (V) drying or partially drying the solution to form a film, 5. A collagen-based, ultraviolet radiation crosslinked, film, 10 membrane, or wafer; wafer, or membrane delivery system comprising at least one (vi) exposing the film, membrane, or wafer to ultraviolet layer of glutaric anhydride derivatized collagen and at least (“UV) radiation in a low oxygen atmosphere for from about 150 lug of Latanoprost, wherein the film, wafer, or about 17 to about 19 minutes; and membrane has been crosslinked with ultraviolet radiation for (vii) recovering the UV-crosslinked, drug-containing, film, from 17 to 19 minutes. 15 membrane, or wafer, wherein the film, membrane, or 6. The collagen-based film, wafer, or membrane of claim 5, wafer is able to provide sustained release of the active wherein the film, wafer, or membrane provides sustained drug at therapeutic levels for at least three months. and/or controlled release of about 1.5ug Latanoprost per day 12. The method of claim 11, wherein the ultraviolet radia for at least about 6 months but for less than one year. tion exposure in step (vii) is for about 18 minutes. 7. The film, wafer, or membrane delivery system of claim.5, 13. The method of claim 11, wherein the method further wherein the film, wafer, or membrane comprises at least comprises treating the UV-crosslinked collagen construct an about 300 ug Latanoprost to provide an effective dosage of aqueous solution for about 1, 2, 3, 4, 5, 6, or 7 days. about 1.5ug per day for at least about 180 days of sustained 14. The method of claim 11, wherein the drug is Latano and/or controlled delivery. prost in an amount sufficient to release an effective dosage of 8. The collagen-based film, wafer, or membrane of claim 5, 25 about 1.5ug per day for at least about 180 days. wherein the Latanoprost present in the film, wafer, or mem 15. The method of claim 11, wherein the method employs brane is resistant to removal by a 1 day treatment in an partial drying of the collagen-drug solution in step (vi) and aqueous buffer. wherein the method further comprises applying a barrier 9. The collagen-based film, wafer, or membrane of claim8, layer comprising collagen derivatized by an acylation agent wherein the Latanoprost present in the film, wafer, or mem 30 that imparts a barrier to diffusion of the drug to one side of the brane is resistant to removal by a 7 day treatment in an film, wafer, or membrane prior to ultraviolet radiation step aqueous buffer. (vii). 10. The film, wafer, or membrane of any one of claims 5 to 16. A collagen-based film or membrane prepared by the 9, further comprising a barrier layer of collagen derivatized method of any one of claims 11 to 15. by an acylation agent. 35 17. A method of treating glaucoma comprising administer 11. A method for preparing a collagen-based film, wafer, or ing to the eye of a glaucoma patient the collagen-based, membrane construct comprising a therapeutic drug and ultraviolet radiation crosslinked, film, wafer, or membrane capable of providing sustained and/or controlled release of delivery system comprising Latanoprost of any one of claims that drug, the method comprising: 5 to 10. (i) preparing derivatized collagen;